Difference between revisions of "Team:Mingdao/Part Collection"

(Prototype team page)
 
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{{Mingdao}}
 
 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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        margin: 0;
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1> Part Collection </h1>
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<p>Did your team make a lot of great parts? Is there a theme that ties all your parts together? Do you have more than 10 parts in this collection? Did you make a CRISPR collection, a MoClo collection, or a collection of awesome pigment parts? Describe your parts collection on this page, so the judges can evaluate you for the Best Part Collection award.</p>
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<p>
 
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While you should put all the characterization information for your parts on the Registry, you are encouraged to explain how all your parts form a collection on this page.
 
−
</p>
 
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<br>
 
−
<h3>Best Part Collection Special Prize</h3>
 
−
<p>Did your team make a lot of great parts? Is there a team that ties all your parts together? Do you have more than 10 parts in this collection? Did you make a CRISPR collection, a MoClo collection or a collection of awesome pigment parts? Tell the judges you should be evaluated for the Best Parts Collection award! To be eligible for this award, these parts must adhere to <a href="http://parts.igem.org/DNA_Submission">Registry sample submission guidelines</a> and have been sent to the Registry of Standard Biological Parts.
 
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<br><br>
 
−
If you have a collection of parts you wish to nominate your team for this <a href="https://2017.igem.org/Judging/Awards">special prize</a>, make sure you add your part numbers to your <a href="https://2017.igem.org/Judging/Judging_Form">judging form</a> and delete the box at the top of this page.</p>
 
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<br>
 
  
  
  
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<h4>Note</h4>
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<p>This page should list all the parts in the collection your team made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page.</p>
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.\36 u\28xlarge\29, .\36 u\24\28xlarge\29 {
 +
width: 50%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\35 u\28xlarge\29, .\35 u\24\28xlarge\29 {
 +
width: 41.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\34 u\28xlarge\29, .\34 u\24\28xlarge\29 {
 +
width: 33.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\33 u\28xlarge\29, .\33 u\24\28xlarge\29 {
 +
width: 25%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\32 u\28xlarge\29, .\32 u\24\28xlarge\29 {
 +
width: 16.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 u\28xlarge\29, .\31 u\24\28xlarge\29 {
 +
width: 8.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 2u\24\28xlarge\29 + *,
 +
.\31 1u\24\28xlarge\29 + *,
 +
.\31 0u\24\28xlarge\29 + *,
 +
.\39 u\24\28xlarge\29 + *,
 +
.\38 u\24\28xlarge\29 + *,
 +
.\37 u\24\28xlarge\29 + *,
 +
.\36 u\24\28xlarge\29 + *,
 +
.\35 u\24\28xlarge\29 + *,
 +
.\34 u\24\28xlarge\29 + *,
 +
.\33 u\24\28xlarge\29 + *,
 +
.\32 u\24\28xlarge\29 + *,
 +
.\31 u\24\28xlarge\29 + * {
 +
clear: left;
 +
}
 +
 +
.\-11u\28xlarge\29 {
 +
margin-left: 91.66667%;
 +
}
 +
 +
.\-10u\28xlarge\29 {
 +
margin-left: 83.33333%;
 +
}
 +
 +
.\-9u\28xlarge\29 {
 +
margin-left: 75%;
 +
}
 +
 +
.\-8u\28xlarge\29 {
 +
margin-left: 66.66667%;
 +
}
 +
 +
.\-7u\28xlarge\29 {
 +
margin-left: 58.33333%;
 +
}
 +
 +
.\-6u\28xlarge\29 {
 +
margin-left: 50%;
 +
}
 +
 +
.\-5u\28xlarge\29 {
 +
margin-left: 41.66667%;
 +
}
 +
 +
.\-4u\28xlarge\29 {
 +
margin-left: 33.33333%;
 +
}
 +
 +
.\-3u\28xlarge\29 {
 +
margin-left: 25%;
 +
}
 +
 +
.\-2u\28xlarge\29 {
 +
margin-left: 16.66667%;
 +
}
 +
 +
.\-1u\28xlarge\29 {
 +
margin-left: 8.33333%;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 1280px) {
 +
 +
.row > * {
 +
padding: 0 0 0 1.5rem;
 +
}
 +
 +
.row {
 +
margin: 0 0 -1px -1.5rem;
 +
}
 +
 +
.row.uniform > * {
 +
padding: 1.5rem 0 0 1.5rem;
 +
}
 +
 +
.row.uniform {
 +
margin: -1.5rem 0 -1px -1.5rem;
 +
}
 +
 +
.row.\32 00\25 > * {
 +
padding: 0 0 0 3rem;
 +
}
 +
 +
.row.\32 00\25 {
 +
margin: 0 0 -1px -3rem;
 +
}
 +
 +
.row.uniform.\32 00\25 > * {
 +
padding: 3rem 0 0 3rem;
 +
}
 +
 +
.row.uniform.\32 00\25 {
 +
margin: -3rem 0 -1px -3rem;
 +
}
 +
 +
.row.\31 50\25 > * {
 +
padding: 0 0 0 2.25rem;
 +
}
 +
 +
.row.\31 50\25 {
 +
margin: 0 0 -1px -2.25rem;
 +
}
 +
 +
.row.uniform.\31 50\25 > * {
 +
padding: 2.25rem 0 0 2.25rem;
 +
}
 +
 +
.row.uniform.\31 50\25 {
 +
margin: -2.25rem 0 -1px -2.25rem;
 +
}
 +
 +
.row.\35 0\25 > * {
 +
padding: 0 0 0 0.75rem;
 +
}
 +
 +
.row.\35 0\25 {
 +
margin: 0 0 -1px -0.75rem;
 +
}
 +
 +
.row.uniform.\35 0\25 > * {
 +
padding: 0.75rem 0 0 0.75rem;
 +
}
 +
 +
.row.uniform.\35 0\25 {
 +
margin: -0.75rem 0 -1px -0.75rem;
 +
}
 +
 +
.row.\32 5\25 > * {
 +
padding: 0 0 0 0.375rem;
 +
}
 +
 +
.row.\32 5\25 {
 +
margin: 0 0 -1px -0.375rem;
 +
}
 +
 +
.row.uniform.\32 5\25 > * {
 +
padding: 0.375rem 0 0 0.375rem;
 +
}
 +
 +
.row.uniform.\32 5\25 {
 +
margin: -0.375rem 0 -1px -0.375rem;
 +
}
 +
 +
.\31 2u\28large\29, .\31 2u\24\28large\29 {
 +
width: 100%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 1u\28large\29, .\31 1u\24\28large\29 {
 +
width: 91.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 0u\28large\29, .\31 0u\24\28large\29 {
 +
width: 83.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\39 u\28large\29, .\39 u\24\28large\29 {
 +
width: 75%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\38 u\28large\29, .\38 u\24\28large\29 {
 +
width: 66.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\37 u\28large\29, .\37 u\24\28large\29 {
 +
width: 58.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\36 u\28large\29, .\36 u\24\28large\29 {
 +
width: 50%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\35 u\28large\29, .\35 u\24\28large\29 {
 +
width: 41.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\34 u\28large\29, .\34 u\24\28large\29 {
 +
width: 33.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\33 u\28large\29, .\33 u\24\28large\29 {
 +
width: 25%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\32 u\28large\29, .\32 u\24\28large\29 {
 +
width: 16.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 u\28large\29, .\31 u\24\28large\29 {
 +
width: 8.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 2u\24\28large\29 + *,
 +
.\31 1u\24\28large\29 + *,
 +
.\31 0u\24\28large\29 + *,
 +
.\39 u\24\28large\29 + *,
 +
.\38 u\24\28large\29 + *,
 +
.\37 u\24\28large\29 + *,
 +
.\36 u\24\28large\29 + *,
 +
.\35 u\24\28large\29 + *,
 +
.\34 u\24\28large\29 + *,
 +
.\33 u\24\28large\29 + *,
 +
.\32 u\24\28large\29 + *,
 +
.\31 u\24\28large\29 + * {
 +
clear: left;
 +
}
 +
 +
.\-11u\28large\29 {
 +
margin-left: 91.66667%;
 +
}
 +
 +
.\-10u\28large\29 {
 +
margin-left: 83.33333%;
 +
}
 +
 +
.\-9u\28large\29 {
 +
margin-left: 75%;
 +
}
 +
 +
.\-8u\28large\29 {
 +
margin-left: 66.66667%;
 +
}
 +
 +
.\-7u\28large\29 {
 +
margin-left: 58.33333%;
 +
}
 +
 +
.\-6u\28large\29 {
 +
margin-left: 50%;
 +
}
 +
 +
.\-5u\28large\29 {
 +
margin-left: 41.66667%;
 +
}
 +
 +
.\-4u\28large\29 {
 +
margin-left: 33.33333%;
 +
}
 +
 +
.\-3u\28large\29 {
 +
margin-left: 25%;
 +
}
 +
 +
.\-2u\28large\29 {
 +
margin-left: 16.66667%;
 +
}
 +
 +
.\-1u\28large\29 {
 +
margin-left: 8.33333%;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
.row > * {
 +
padding: 0 0 0 1.5rem;
 +
}
 +
 +
.row {
 +
margin: 0 0 -1px -1.5rem;
 +
}
 +
 +
.row.uniform > * {
 +
padding: 1.5rem 0 0 1.5rem;
 +
}
 +
 +
.row.uniform {
 +
margin: -1.5rem 0 -1px -1.5rem;
 +
}
 +
 +
.row.\32 00\25 > * {
 +
padding: 0 0 0 3rem;
 +
}
 +
 +
.row.\32 00\25 {
 +
margin: 0 0 -1px -3rem;
 +
}
 +
 +
.row.uniform.\32 00\25 > * {
 +
padding: 3rem 0 0 3rem;
 +
}
 +
 +
.row.uniform.\32 00\25 {
 +
margin: -3rem 0 -1px -3rem;
 +
}
 +
 +
.row.\31 50\25 > * {
 +
padding: 0 0 0 2.25rem;
 +
}
 +
 +
.row.\31 50\25 {
 +
margin: 0 0 -1px -2.25rem;
 +
}
 +
 +
.row.uniform.\31 50\25 > * {
 +
padding: 2.25rem 0 0 2.25rem;
 +
}
 +
 +
.row.uniform.\31 50\25 {
 +
margin: -2.25rem 0 -1px -2.25rem;
 +
}
 +
 +
.row.\35 0\25 > * {
 +
padding: 0 0 0 0.75rem;
 +
}
 +
 +
.row.\35 0\25 {
 +
margin: 0 0 -1px -0.75rem;
 +
}
 +
 +
.row.uniform.\35 0\25 > * {
 +
padding: 0.75rem 0 0 0.75rem;
 +
}
 +
 +
.row.uniform.\35 0\25 {
 +
margin: -0.75rem 0 -1px -0.75rem;
 +
}
 +
 +
.row.\32 5\25 > * {
 +
padding: 0 0 0 0.375rem;
 +
}
 +
 +
.row.\32 5\25 {
 +
margin: 0 0 -1px -0.375rem;
 +
}
 +
 +
.row.uniform.\32 5\25 > * {
 +
padding: 0.375rem 0 0 0.375rem;
 +
}
 +
 +
.row.uniform.\32 5\25 {
 +
margin: -0.375rem 0 -1px -0.375rem;
 +
}
 +
 +
.\31 2u\28medium\29, .\31 2u\24\28medium\29 {
 +
width: 100%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 1u\28medium\29, .\31 1u\24\28medium\29 {
 +
width: 91.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 0u\28medium\29, .\31 0u\24\28medium\29 {
 +
width: 83.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\39 u\28medium\29, .\39 u\24\28medium\29 {
 +
width: 75%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\38 u\28medium\29, .\38 u\24\28medium\29 {
 +
width: 66.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\37 u\28medium\29, .\37 u\24\28medium\29 {
 +
width: 58.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\36 u\28medium\29, .\36 u\24\28medium\29 {
 +
width: 50%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\35 u\28medium\29, .\35 u\24\28medium\29 {
 +
width: 41.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\34 u\28medium\29, .\34 u\24\28medium\29 {
 +
width: 33.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\33 u\28medium\29, .\33 u\24\28medium\29 {
 +
width: 25%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\32 u\28medium\29, .\32 u\24\28medium\29 {
 +
width: 16.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 u\28medium\29, .\31 u\24\28medium\29 {
 +
width: 8.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 2u\24\28medium\29 + *,
 +
.\31 1u\24\28medium\29 + *,
 +
.\31 0u\24\28medium\29 + *,
 +
.\39 u\24\28medium\29 + *,
 +
.\38 u\24\28medium\29 + *,
 +
.\37 u\24\28medium\29 + *,
 +
.\36 u\24\28medium\29 + *,
 +
.\35 u\24\28medium\29 + *,
 +
.\34 u\24\28medium\29 + *,
 +
.\33 u\24\28medium\29 + *,
 +
.\32 u\24\28medium\29 + *,
 +
.\31 u\24\28medium\29 + * {
 +
clear: left;
 +
}
 +
 +
.\-11u\28medium\29 {
 +
margin-left: 91.66667%;
 +
}
 +
 +
.\-10u\28medium\29 {
 +
margin-left: 83.33333%;
 +
}
 +
 +
.\-9u\28medium\29 {
 +
margin-left: 75%;
 +
}
 +
 +
.\-8u\28medium\29 {
 +
margin-left: 66.66667%;
 +
}
 +
 +
.\-7u\28medium\29 {
 +
margin-left: 58.33333%;
 +
}
 +
 +
.\-6u\28medium\29 {
 +
margin-left: 50%;
 +
}
 +
 +
.\-5u\28medium\29 {
 +
margin-left: 41.66667%;
 +
}
 +
 +
.\-4u\28medium\29 {
 +
margin-left: 33.33333%;
 +
}
 +
 +
.\-3u\28medium\29 {
 +
margin-left: 25%;
 +
}
 +
 +
.\-2u\28medium\29 {
 +
margin-left: 16.66667%;
 +
}
 +
 +
.\-1u\28medium\29 {
 +
margin-left: 8.33333%;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
.row > * {
 +
padding: 0 0 0 1.25rem;
 +
}
 +
 +
.row {
 +
margin: 0 0 -1px -1.25rem;
 +
}
 +
 +
.row.uniform > * {
 +
padding: 1.25rem 0 0 1.25rem;
 +
}
 +
 +
.row.uniform {
 +
margin: -1.25rem 0 -1px -1.25rem;
 +
}
 +
 +
.row.\32 00\25 > * {
 +
padding: 0 0 0 2.5rem;
 +
}
 +
 +
.row.\32 00\25 {
 +
margin: 0 0 -1px -2.5rem;
 +
}
 +
 +
.row.uniform.\32 00\25 > * {
 +
padding: 2.5rem 0 0 2.5rem;
 +
}
 +
 +
.row.uniform.\32 00\25 {
 +
margin: -2.5rem 0 -1px -2.5rem;
 +
}
 +
 +
.row.\31 50\25 > * {
 +
padding: 0 0 0 1.875rem;
 +
}
 +
 +
.row.\31 50\25 {
 +
margin: 0 0 -1px -1.875rem;
 +
}
 +
 +
.row.uniform.\31 50\25 > * {
 +
padding: 1.875rem 0 0 1.875rem;
 +
}
 +
 +
.row.uniform.\31 50\25 {
 +
margin: -1.875rem 0 -1px -1.875rem;
 +
}
 +
 +
.row.\35 0\25 > * {
 +
padding: 0 0 0 0.625rem;
 +
}
 +
 +
.row.\35 0\25 {
 +
margin: 0 0 -1px -0.625rem;
 +
}
 +
 +
.row.uniform.\35 0\25 > * {
 +
padding: 0.625rem 0 0 0.625rem;
 +
}
 +
 +
.row.uniform.\35 0\25 {
 +
margin: -0.625rem 0 -1px -0.625rem;
 +
}
 +
 +
.row.\32 5\25 > * {
 +
padding: 0 0 0 0.3125rem;
 +
}
 +
 +
.row.\32 5\25 {
 +
margin: 0 0 -1px -0.3125rem;
 +
}
 +
 +
.row.uniform.\32 5\25 > * {
 +
padding: 0.3125rem 0 0 0.3125rem;
 +
}
 +
 +
.row.uniform.\32 5\25 {
 +
margin: -0.3125rem 0 -1px -0.3125rem;
 +
}
 +
 +
.\31 2u\28small\29, .\31 2u\24\28small\29 {
 +
width: 100%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 1u\28small\29, .\31 1u\24\28small\29 {
 +
width: 91.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 0u\28small\29, .\31 0u\24\28small\29 {
 +
width: 83.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\39 u\28small\29, .\39 u\24\28small\29 {
 +
width: 75%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\38 u\28small\29, .\38 u\24\28small\29 {
 +
width: 66.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\37 u\28small\29, .\37 u\24\28small\29 {
 +
width: 58.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\36 u\28small\29, .\36 u\24\28small\29 {
 +
width: 50%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\35 u\28small\29, .\35 u\24\28small\29 {
 +
width: 41.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\34 u\28small\29, .\34 u\24\28small\29 {
 +
width: 33.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\33 u\28small\29, .\33 u\24\28small\29 {
 +
width: 25%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\32 u\28small\29, .\32 u\24\28small\29 {
 +
width: 16.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 u\28small\29, .\31 u\24\28small\29 {
 +
width: 8.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 2u\24\28small\29 + *,
 +
.\31 1u\24\28small\29 + *,
 +
.\31 0u\24\28small\29 + *,
 +
.\39 u\24\28small\29 + *,
 +
.\38 u\24\28small\29 + *,
 +
.\37 u\24\28small\29 + *,
 +
.\36 u\24\28small\29 + *,
 +
.\35 u\24\28small\29 + *,
 +
.\34 u\24\28small\29 + *,
 +
.\33 u\24\28small\29 + *,
 +
.\32 u\24\28small\29 + *,
 +
.\31 u\24\28small\29 + * {
 +
clear: left;
 +
}
 +
 +
.\-11u\28small\29 {
 +
margin-left: 91.66667%;
 +
}
 +
 +
.\-10u\28small\29 {
 +
margin-left: 83.33333%;
 +
}
 +
 +
.\-9u\28small\29 {
 +
margin-left: 75%;
 +
}
 +
 +
.\-8u\28small\29 {
 +
margin-left: 66.66667%;
 +
}
 +
 +
.\-7u\28small\29 {
 +
margin-left: 58.33333%;
 +
}
 +
 +
.\-6u\28small\29 {
 +
margin-left: 50%;
 +
}
 +
 +
.\-5u\28small\29 {
 +
margin-left: 41.66667%;
 +
}
 +
 +
.\-4u\28small\29 {
 +
margin-left: 33.33333%;
 +
}
 +
 +
.\-3u\28small\29 {
 +
margin-left: 25%;
 +
}
 +
 +
.\-2u\28small\29 {
 +
margin-left: 16.66667%;
 +
}
 +
 +
.\-1u\28small\29 {
 +
margin-left: 8.33333%;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 480px) {
 +
 +
.row > * {
 +
padding: 0 0 0 1.25rem;
 +
}
 +
 +
.row {
 +
margin: 0 0 -1px -1.25rem;
 +
}
 +
 +
.row.uniform > * {
 +
padding: 1.25rem 0 0 1.25rem;
 +
}
 +
 +
.row.uniform {
 +
margin: -1.25rem 0 -1px -1.25rem;
 +
}
 +
 +
.row.\32 00\25 > * {
 +
padding: 0 0 0 2.5rem;
 +
}
 +
 +
.row.\32 00\25 {
 +
margin: 0 0 -1px -2.5rem;
 +
}
 +
 +
.row.uniform.\32 00\25 > * {
 +
padding: 2.5rem 0 0 2.5rem;
 +
}
 +
 +
.row.uniform.\32 00\25 {
 +
margin: -2.5rem 0 -1px -2.5rem;
 +
}
 +
 +
.row.\31 50\25 > * {
 +
padding: 0 0 0 1.875rem;
 +
}
 +
 +
.row.\31 50\25 {
 +
margin: 0 0 -1px -1.875rem;
 +
}
 +
 +
.row.uniform.\31 50\25 > * {
 +
padding: 1.875rem 0 0 1.875rem;
 +
}
 +
 +
.row.uniform.\31 50\25 {
 +
margin: -1.875rem 0 -1px -1.875rem;
 +
}
 +
 +
.row.\35 0\25 > * {
 +
padding: 0 0 0 0.625rem;
 +
}
 +
 +
.row.\35 0\25 {
 +
margin: 0 0 -1px -0.625rem;
 +
}
 +
 +
.row.uniform.\35 0\25 > * {
 +
padding: 0.625rem 0 0 0.625rem;
 +
}
 +
 +
.row.uniform.\35 0\25 {
 +
margin: -0.625rem 0 -1px -0.625rem;
 +
}
 +
 +
.row.\32 5\25 > * {
 +
padding: 0 0 0 0.3125rem;
 +
}
 +
 +
.row.\32 5\25 {
 +
margin: 0 0 -1px -0.3125rem;
 +
}
 +
 +
.row.uniform.\32 5\25 > * {
 +
padding: 0.3125rem 0 0 0.3125rem;
 +
}
 +
 +
.row.uniform.\32 5\25 {
 +
margin: -0.3125rem 0 -1px -0.3125rem;
 +
}
 +
 +
.\31 2u\28xsmall\29, .\31 2u\24\28xsmall\29 {
 +
width: 100%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 1u\28xsmall\29, .\31 1u\24\28xsmall\29 {
 +
width: 91.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 0u\28xsmall\29, .\31 0u\24\28xsmall\29 {
 +
width: 83.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\39 u\28xsmall\29, .\39 u\24\28xsmall\29 {
 +
width: 75%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\38 u\28xsmall\29, .\38 u\24\28xsmall\29 {
 +
width: 66.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\37 u\28xsmall\29, .\37 u\24\28xsmall\29 {
 +
width: 58.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\36 u\28xsmall\29, .\36 u\24\28xsmall\29 {
 +
width: 50%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\35 u\28xsmall\29, .\35 u\24\28xsmall\29 {
 +
width: 41.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\34 u\28xsmall\29, .\34 u\24\28xsmall\29 {
 +
width: 33.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\33 u\28xsmall\29, .\33 u\24\28xsmall\29 {
 +
width: 25%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\32 u\28xsmall\29, .\32 u\24\28xsmall\29 {
 +
width: 16.6666666667%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 u\28xsmall\29, .\31 u\24\28xsmall\29 {
 +
width: 8.3333333333%;
 +
clear: none;
 +
margin-left: 0;
 +
}
 +
 +
.\31 2u\24\28xsmall\29 + *,
 +
.\31 1u\24\28xsmall\29 + *,
 +
.\31 0u\24\28xsmall\29 + *,
 +
.\39 u\24\28xsmall\29 + *,
 +
.\38 u\24\28xsmall\29 + *,
 +
.\37 u\24\28xsmall\29 + *,
 +
.\36 u\24\28xsmall\29 + *,
 +
.\35 u\24\28xsmall\29 + *,
 +
.\34 u\24\28xsmall\29 + *,
 +
.\33 u\24\28xsmall\29 + *,
 +
.\32 u\24\28xsmall\29 + *,
 +
.\31 u\24\28xsmall\29 + * {
 +
clear: left;
 +
}
 +
 +
.\-11u\28xsmall\29 {
 +
margin-left: 91.66667%;
 +
}
 +
 +
.\-10u\28xsmall\29 {
 +
margin-left: 83.33333%;
 +
}
 +
 +
.\-9u\28xsmall\29 {
 +
margin-left: 75%;
 +
}
 +
 +
.\-8u\28xsmall\29 {
 +
margin-left: 66.66667%;
 +
}
 +
 +
.\-7u\28xsmall\29 {
 +
margin-left: 58.33333%;
 +
}
 +
 +
.\-6u\28xsmall\29 {
 +
margin-left: 50%;
 +
}
 +
 +
.\-5u\28xsmall\29 {
 +
margin-left: 41.66667%;
 +
}
 +
 +
.\-4u\28xsmall\29 {
 +
margin-left: 33.33333%;
 +
}
 +
 +
.\-3u\28xsmall\29 {
 +
margin-left: 25%;
 +
}
 +
 +
.\-2u\28xsmall\29 {
 +
margin-left: 16.66667%;
 +
}
 +
 +
.\-1u\28xsmall\29 {
 +
margin-left: 8.33333%;
 +
}
 +
 +
}
 +
 +
/* Basic */
 +
 +
@-ms-viewport {
 +
width: device-width;
 +
}
 +
 +
body {
 +
-ms-overflow-style: scrollbar;
 +
}
 +
 +
@media screen and (max-width: 480px) {
 +
 +
html, body {
 +
min-width: 320px;
 +
}
 +
 +
}
 +
 +
body {
 +
background: #fff;
 +
}
 +
 +
body.is-loading *, body.is-loading *:before, body.is-loading *:after {
 +
-moz-animation: none !important;
 +
-webkit-animation: none !important;
 +
-ms-animation: none !important;
 +
animation: none !important;
 +
-moz-transition: none !important;
 +
-webkit-transition: none !important;
 +
-ms-transition: none !important;
 +
transition: none !important;
 +
}
 +
 +
/* Type */
 +
 +
html {
 +
font-size: 13pt;
 +
}
 +
 +
@media screen and (max-width: 1680px) {
 +
 +
html {
 +
font-size: 11pt;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 1280px) {
 +
 +
html {
 +
font-size: 11pt;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
html {
 +
font-size: 12pt;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
html {
 +
font-size: 12pt;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 480px) {
 +
 +
html {
 +
font-size: 12pt;
 +
}
 +
 +
}
 +
 +
body {
 +
background-color: #fff;
 +
color: #444;
 +
}
 +
 +
body, input, select, textarea {
 +
font-family: "Poppins", sans-serif;
 +
font-weight: 300;
 +
font-size: 1rem;
 +
line-height: 1.65;
 +
}
 +
 +
a {
 +
text-decoration: underline;
 +
}
 +
 +
a:hover {
 +
text-decoration: none;
 +
}
 +
 +
strong, b {
 +
font-weight: 600;
 +
}
 +
 +
em, i {
 +
font-style: italic;
 +
}
 +
 +
p {
 +
margin: 0 0 2rem 0;
 +
}
 +
 +
p.special {
 +
text-transform: uppercase;
 +
font-size: .75rem;
 +
font-weight: 300;
 +
margin: 0 0 .5rem 0;
 +
padding: 0 0 1rem 0;
 +
letter-spacing: .25rem;
 +
}
 +
 +
p.special:after {
 +
content: '';
 +
position: absolute;
 +
margin: auto;
 +
right: 0;
 +
bottom: 0;
 +
left: 0;
 +
width: 50%;
 +
height: 1px;
 +
background-color: rgba(0, 0, 0, 0.125);
 +
}
 +
 +
h1, h2, h3, h4, h5, h6 {
 +
font-weight: 300;
 +
line-height: 1.5;
 +
margin: 0 0 1rem 0;
 +
}
 +
 +
h1 a, h2 a, h3 a, h4 a, h5 a, h6 a {
 +
color: inherit;
 +
text-decoration: none;
 +
}
 +
 +
h2 {
 +
font-size: 1.75rem;
 +
}
 +
 +
h3 {
 +
font-size: 1.35rem;
 +
}
 +
 +
h4 {
 +
font-size: 1.1rem;
 +
}
 +
 +
h5 {
 +
font-size: 0.9rem;
 +
}
 +
 +
h6 {
 +
font-size: 0.7rem;
 +
}
 +
 +
sub {
 +
font-size: 0.8rem;
 +
position: relative;
 +
top: 0.5rem;
 +
}
 +
 +
sup {
 +
font-size: 0.8rem;
 +
position: relative;
 +
top: -0.5rem;
 +
}
 +
 +
blockquote {
 +
border-left: solid 4px;
 +
font-style: italic;
 +
margin: 0 0 2rem 0;
 +
padding: 0.5rem 0 0.5rem 2rem;
 +
}
 +
 +
code {
 +
border-radius: 2px;
 +
border: solid 1px;
 +
font-family: "Courier New", monospace;
 +
font-size: 0.9rem;
 +
margin: 0 0.25rem;
 +
padding: 0.25rem 0.65rem;
 +
}
 +
 +
pre {
 +
-webkit-overflow-scrolling: touch;
 +
font-family: "Courier New", monospace;
 +
font-size: 0.9rem;
 +
margin: 0 0 2rem 0;
 +
}
 +
 +
pre code {
 +
display: block;
 +
line-height: 1.75;
 +
padding: 1rem 1.5rem;
 +
overflow-x: auto;
 +
}
 +
 +
hr {
 +
border: 0;
 +
border-bottom: solid 1px;
 +
margin: 2rem 0;
 +
}
 +
 +
hr.major {
 +
margin: 3rem 0;
 +
}
 +
 +
.align-left {
 +
text-align: left;
 +
}
 +
 +
.align-center {
 +
text-align: center;
 +
}
 +
 +
.align-right {
 +
text-align: right;
 +
}
 +
 +
input, select, textarea {
 +
color: #555;
 +
}
 +
 +
a {
 +
color: #8a4680;
 +
}
 +
 +
strong, b {
 +
color: #555;
 +
}
 +
 +
h1, h2, h3, h4, h5, h6 {
 +
color: #555;
 +
}
 +
 +
blockquote {
 +
border-left-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
code {
 +
background: rgba(144, 144, 144, 0.075);
 +
border-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
hr {
 +
border-bottom-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
/* Box */
 +
 +
.box {
 +
margin-bottom: 2rem;
 +
background: #FFF;
 +
}
 +
 +
.box .image.fit {
 +
margin: 0;
 +
border-radius: 0;
 +
}
 +
 +
.box .image.fit img {
 +
border-radius: 0;
 +
}
 +
 +
.box header h2 {
 +
margin-bottom: 2rem;
 +
}
 +
 +
.box header p {
 +
text-transform: uppercase;
 +
font-size: .75rem;
 +
font-weight: 300;
 +
margin: 0 0 .25rem 0;
 +
padding: 0 0 .75rem 0;
 +
letter-spacing: .25rem;
 +
}
 +
 +
.box header p:after {
 +
content: '';
 +
position: absolute;
 +
margin: auto;
 +
right: 0;
 +
bottom: 0;
 +
left: 0;
 +
width: 50%;
 +
height: 1px;
 +
background-color: rgba(0, 0, 0, 0.125);
 +
}
 +
 +
.box .content {
 +
padding: 3rem;
 +
}
 +
 +
.box > :last-child,
 +
.box > :last-child > :last-child,
 +
.box > :last-child > :last-child > :last-child {
 +
margin-bottom: 0;
 +
}
 +
 +
.box.alt {
 +
border: 0;
 +
border-radius: 0;
 +
padding: 0;
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
.box .content {
 +
padding: 2rem;
 +
}
 +
 +
}
 +
 +
.box {
 +
border-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
/* Button */
 +
 +
input[type="submit"],
 +
input[type="reset"],
 +
input[type="button"],
 +
button,
 +
.button {
 +
-moz-appearance: none;
 +
-webkit-appearance: none;
 +
-ms-appearance: none;
 +
appearance: none;
 +
-moz-transition: background-color 0.2s ease-in-out, color 0.2s ease-in-out;
 +
-webkit-transition: background-color 0.2s ease-in-out, color 0.2s ease-in-out;
 +
-ms-transition: background-color 0.2s ease-in-out, color 0.2s ease-in-out;
 +
transition: background-color 0.2s ease-in-out, color 0.2s ease-in-out;
 +
border-radius: 2px;
 +
border: 0;
 +
cursor: pointer;
 +
display: inline-block;
 +
font-weight: 300;
 +
height: 2.85rem;
 +
line-height: 2.95rem;
 +
padding: 0 1.5rem;
 +
text-align: center;
 +
text-decoration: none;
 +
text-transform: uppercase;
 +
white-space: nowrap;
 +
}
 +
 +
input[type="submit"].icon,
 +
input[type="reset"].icon,
 +
input[type="button"].icon,
 +
button.icon,
 +
.button.icon {
 +
padding-left: 1.35rem;
 +
}
 +
 +
input[type="submit"].icon:before,
 +
input[type="reset"].icon:before,
 +
input[type="button"].icon:before,
 +
button.icon:before,
 +
.button.icon:before {
 +
margin-right: 0.5rem;
 +
}
 +
 +
input[type="submit"].fit,
 +
input[type="reset"].fit,
 +
input[type="button"].fit,
 +
button.fit,
 +
.button.fit {
 +
display: block;
 +
margin: 0 0 1rem 0;
 +
width: 100%;
 +
}
 +
 +
input[type="submit"].small,
 +
input[type="reset"].small,
 +
input[type="button"].small,
 +
button.small,
 +
.button.small {
 +
font-size: 0.8rem;
 +
}
 +
 +
input[type="submit"].big,
 +
input[type="reset"].big,
 +
input[type="button"].big,
 +
button.big,
 +
.button.big {
 +
font-size: 1.35rem;
 +
}
 +
 +
input[type="submit"].disabled, input[type="submit"]:disabled,
 +
input[type="reset"].disabled,
 +
input[type="reset"]:disabled,
 +
input[type="button"].disabled,
 +
input[type="button"]:disabled,
 +
button.disabled,
 +
button:disabled,
 +
.button.disabled,
 +
.button:disabled {
 +
-moz-pointer-events: none;
 +
-webkit-pointer-events: none;
 +
-ms-pointer-events: none;
 +
pointer-events: none;
 +
opacity: 0.25;
 +
}
 +
 +
@media screen and (max-width: 480px) {
 +
 +
input[type="submit"],
 +
input[type="reset"],
 +
input[type="button"],
 +
button,
 +
.button {
 +
padding: 0;
 +
width: 100%;
 +
}
 +
 +
}
 +
 +
input[type="submit"],
 +
input[type="reset"],
 +
input[type="button"],
 +
button,
 +
.button {
 +
background-color: #f2f2f2;
 +
color: #000 !important;
 +
}
 +
 +
input[type="submit"]:hover,
 +
input[type="reset"]:hover,
 +
input[type="button"]:hover,
 +
button:hover,
 +
.button:hover {
 +
background-color: white;
 +
}
 +
 +
input[type="submit"]:active,
 +
input[type="reset"]:active,
 +
input[type="button"]:active,
 +
button:active,
 +
.button:active {
 +
background-color: #e5e5e5;
 +
}
 +
 +
input[type="submit"].alt,
 +
input[type="reset"].alt,
 +
input[type="button"].alt,
 +
button.alt,
 +
.button.alt {
 +
background-color: transparent;
 +
box-shadow: inset 0 0 0 2px rgba(144, 144, 144, 0.25);
 +
color: #555 !important;
 +
}
 +
 +
input[type="submit"].alt:hover,
 +
input[type="reset"].alt:hover,
 +
input[type="button"].alt:hover,
 +
button.alt:hover,
 +
.button.alt:hover {
 +
background-color: rgba(144, 144, 144, 0.075);
 +
}
 +
 +
input[type="submit"].alt:active,
 +
input[type="reset"].alt:active,
 +
input[type="button"].alt:active,
 +
button.alt:active,
 +
.button.alt:active {
 +
background-color: rgba(144, 144, 144, 0.2);
 +
}
 +
 +
input[type="submit"].alt.icon:before,
 +
input[type="reset"].alt.icon:before,
 +
input[type="button"].alt.icon:before,
 +
button.alt.icon:before,
 +
.button.alt.icon:before {
 +
color: #bbb;
 +
}
 +
 +
input[type="submit"].special,
 +
input[type="reset"].special,
 +
input[type="button"].special,
 +
button.special,
 +
.button.special {
 +
background-color: #8a4680;
 +
color: #ffffff !important;
 +
}
 +
 +
input[type="submit"].special:hover,
 +
input[type="reset"].special:hover,
 +
input[type="button"].special:hover,
 +
button.special:hover,
 +
.button.special:hover {
 +
background-color: #9b4f90;
 +
}
 +
 +
input[type="submit"].special:active,
 +
input[type="reset"].special:active,
 +
input[type="button"].special:active,
 +
button.special:active,
 +
.button.special:active {
 +
background-color: #793d70;
 +
}
 +
 +
/* Form */
 +
 +
form {
 +
margin: 0 0 2rem 0;
 +
}
 +
 +
label {
 +
display: block;
 +
font-size: 0.9rem;
 +
font-weight: 700;
 +
margin: 0 0 1rem 0;
 +
}
 +
 +
input[type="text"],
 +
input[type="password"],
 +
input[type="email"],
 +
select,
 +
textarea {
 +
-moz-appearance: none;
 +
-webkit-appearance: none;
 +
-ms-appearance: none;
 +
appearance: none;
 +
border-radius: 2px;
 +
border: none;
 +
border: solid 1px;
 +
color: inherit;
 +
display: block;
 +
outline: 0;
 +
padding: 0 1rem;
 +
text-decoration: none;
 +
width: 100%;
 +
}
 +
 +
input[type="text"]:invalid,
 +
input[type="password"]:invalid,
 +
input[type="email"]:invalid,
 +
select:invalid,
 +
textarea:invalid {
 +
box-shadow: none;
 +
}
 +
 +
.select-wrapper {
 +
text-decoration: none;
 +
display: block;
 +
position: relative;
 +
}
 +
 +
.select-wrapper:before {
 +
-moz-osx-font-smoothing: grayscale;
 +
-webkit-font-smoothing: antialiased;
 +
font-family: FontAwesome;
 +
font-style: normal;
 +
font-weight: normal;
 +
text-transform: none !important;
 +
}
 +
 +
.select-wrapper:before {
 +
content: '\f078';
 +
display: block;
 +
height: 2.75rem;
 +
line-height: 2.75rem;
 +
pointer-events: none;
 +
position: absolute;
 +
right: 0;
 +
text-align: center;
 +
top: 0;
 +
width: 2.75rem;
 +
}
 +
 +
.select-wrapper select::-ms-expand {
 +
display: none;
 +
}
 +
 +
input[type="text"],
 +
input[type="password"],
 +
input[type="email"],
 +
select {
 +
height: 2.75rem;
 +
}
 +
 +
textarea {
 +
padding: 0.75rem 1rem;
 +
}
 +
 +
input[type="checkbox"],
 +
input[type="radio"] {
 +
-moz-appearance: none;
 +
-webkit-appearance: none;
 +
-ms-appearance: none;
 +
appearance: none;
 +
display: block;
 +
float: left;
 +
margin-right: -2rem;
 +
opacity: 0;
 +
width: 1rem;
 +
z-index: -1;
 +
}
 +
 +
input[type="checkbox"] + label,
 +
input[type="radio"] + label {
 +
text-decoration: none;
 +
cursor: pointer;
 +
display: inline-block;
 +
font-size: 1rem;
 +
font-weight: 400;
 +
padding-left: 2.4rem;
 +
padding-right: 0.75rem;
 +
position: relative;
 +
}
 +
 +
input[type="checkbox"] + label:before,
 +
input[type="radio"] + label:before {
 +
-moz-osx-font-smoothing: grayscale;
 +
-webkit-font-smoothing: antialiased;
 +
font-family: FontAwesome;
 +
font-style: normal;
 +
font-weight: normal;
 +
text-transform: none !important;
 +
}
 +
 +
input[type="checkbox"] + label:before,
 +
input[type="radio"] + label:before {
 +
border-radius: 2px;
 +
border: solid 1px;
 +
content: '';
 +
display: inline-block;
 +
height: 1.65rem;
 +
left: 0;
 +
line-height: 1.58125rem;
 +
position: absolute;
 +
text-align: center;
 +
top: 0;
 +
width: 1.65rem;
 +
}
 +
 +
input[type="checkbox"]:checked + label:before,
 +
input[type="radio"]:checked + label:before {
 +
content: '\f00c';
 +
}
 +
 +
input[type="checkbox"] + label:before {
 +
border-radius: 2px;
 +
}
 +
 +
input[type="radio"] + label:before {
 +
border-radius: 100%;
 +
}
 +
 +
::-webkit-input-placeholder {
 +
opacity: 1.0;
 +
}
 +
 +
:-moz-placeholder {
 +
opacity: 1.0;
 +
}
 +
 +
::-moz-placeholder {
 +
opacity: 1.0;
 +
}
 +
 +
:-ms-input-placeholder {
 +
opacity: 1.0;
 +
}
 +
 +
.formerize-placeholder {
 +
opacity: 1.0;
 +
}
 +
 +
label {
 +
color: #555;
 +
}
 +
 +
input[type="text"],
 +
input[type="password"],
 +
input[type="email"],
 +
select,
 +
textarea {
 +
background: rgba(144, 144, 144, 0.075);
 +
border-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
input[type="text"]:focus,
 +
input[type="password"]:focus,
 +
input[type="email"]:focus,
 +
select:focus,
 +
textarea:focus {
 +
border-color: #8a4680;
 +
box-shadow: 0 0 0 1px #8a4680;
 +
}
 +
 +
.select-wrapper:before {
 +
color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
input[type="checkbox"] + label,
 +
input[type="radio"] + label {
 +
color: #444;
 +
}
 +
 +
input[type="checkbox"] + label:before,
 +
input[type="radio"] + label:before {
 +
background: rgba(144, 144, 144, 0.075);
 +
border-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
input[type="checkbox"]:checked + label:before,
 +
input[type="radio"]:checked + label:before {
 +
background-color: #8a4680;
 +
border-color: #8a4680;
 +
color: #ffffff;
 +
}
 +
 +
input[type="checkbox"]:focus + label:before,
 +
input[type="radio"]:focus + label:before {
 +
border-color: #8a4680;
 +
box-shadow: 0 0 0 1px #8a4680;
 +
}
 +
 +
::-webkit-input-placeholder {
 +
color: #bbb !important;
 +
}
 +
 +
:-moz-placeholder {
 +
color: #bbb !important;
 +
}
 +
 +
::-moz-placeholder {
 +
color: #bbb !important;
 +
}
 +
 +
:-ms-input-placeholder {
 +
color: #bbb !important;
 +
}
 +
 +
.formerize-placeholder {
 +
color: #bbb !important;
 +
}
 +
 +
/* Icon */
 +
 +
.icon {
 +
text-decoration: none;
 +
border-bottom: none;
 +
position: relative;
 +
}
 +
 +
.icon:before {
 +
-moz-osx-font-smoothing: grayscale;
 +
-webkit-font-smoothing: antialiased;
 +
font-family: FontAwesome;
 +
font-style: normal;
 +
font-weight: normal;
 +
text-transform: none !important;
 +
}
 +
 +
.icon > .label {
 +
display: none;
 +
}
 +
 +
/* Image */
 +
 +
.image {
 +
border-radius: 2px;
 +
border: 0;
 +
display: inline-block;
 +
position: relative;
 +
}
 +
 +
.image img {
 +
border-radius: 2px;
 +
display: block;
 +
}
 +
 +
.image.left, .image.right {
 +
max-width: 40%;
 +
}
 +
 +
.image.left img, .image.right img {
 +
width: 100%;
 +
}
 +
 +
.image.left {
 +
float: left;
 +
margin: 0 1.5rem 1rem 0;
 +
top: 0.25rem;
 +
}
 +
 +
.image.right {
 +
float: right;
 +
margin: 0 0 1rem 1.5rem;
 +
top: 0.25rem;
 +
}
 +
 +
.image.fit {
 +
display: block;
 +
margin: 0 0 2rem 0;
 +
width: 100%;
 +
}
 +
 +
.image.fit img {
 +
width: 100%;
 +
}
 +
 +
.image.main {
 +
display: block;
 +
margin: 0 0 3rem 0;
 +
width: 100%;
 +
}
 +
 +
.image.main img {
 +
width: 100%;
 +
}
 +
 +
/* List */
 +
 +
ol {
 +
list-style: decimal;
 +
margin: 0 0 2rem 0;
 +
padding-left: 1.25rem;
 +
}
 +
 +
ol li {
 +
padding-left: 0.25rem;
 +
}
 +
 +
ul {
 +
list-style: disc;
 +
margin: 0 0 2rem 0;
 +
padding-left: 1rem;
 +
}
 +
 +
ul li {
 +
padding-left: 0.5rem;
 +
}
 +
 +
ul.alt {
 +
list-style: none;
 +
padding-left: 0;
 +
}
 +
 +
ul.alt li {
 +
border-top: solid 1px;
 +
padding: 0.5rem 0;
 +
}
 +
 +
ul.alt li:first-child {
 +
border-top: 0;
 +
padding-top: 0;
 +
}
 +
 +
ul.icons {
 +
cursor: default;
 +
list-style: none;
 +
padding-left: 0;
 +
}
 +
 +
ul.icons li {
 +
display: inline-block;
 +
padding: 0 1rem 0 0;
 +
}
 +
 +
ul.icons li:last-child {
 +
padding-right: 0;
 +
}
 +
 +
ul.icons li .icon:before {
 +
font-size: 2rem;
 +
}
 +
 +
ul.actions {
 +
cursor: default;
 +
list-style: none;
 +
padding-left: 0;
 +
}
 +
 +
ul.actions li {
 +
display: inline-block;
 +
padding: 0 1rem 0 0;
 +
vertical-align: middle;
 +
}
 +
 +
ul.actions li:last-child {
 +
padding-right: 0;
 +
}
 +
 +
ul.actions.small li {
 +
padding: 0 0.5rem 0 0;
 +
}
 +
 +
ul.actions.vertical li {
 +
display: block;
 +
padding: 1rem 0 0 0;
 +
}
 +
 +
ul.actions.vertical li:first-child {
 +
padding-top: 0;
 +
}
 +
 +
ul.actions.vertical li > * {
 +
margin-bottom: 0;
 +
}
 +
 +
ul.actions.vertical.small li {
 +
padding: 0.5rem 0 0 0;
 +
}
 +
 +
ul.actions.vertical.small li:first-child {
 +
padding-top: 0;
 +
}
 +
 +
ul.actions.fit {
 +
display: table;
 +
margin-left: -1rem;
 +
padding: 0;
 +
table-layout: fixed;
 +
width: calc(100% + 1rem);
 +
}
 +
 +
ul.actions.fit li {
 +
display: table-cell;
 +
padding: 0 0 0 1rem;
 +
}
 +
 +
ul.actions.fit li > * {
 +
margin-bottom: 0;
 +
}
 +
 +
ul.actions.fit.small {
 +
margin-left: -0.5rem;
 +
width: calc(100% + 0.5rem);
 +
}
 +
 +
ul.actions.fit.small li {
 +
padding: 0 0 0 0.5rem;
 +
}
 +
 +
@media screen and (max-width: 480px) {
 +
 +
ul.actions {
 +
margin: 0 0 2rem 0;
 +
}
 +
 +
ul.actions li {
 +
padding: 1rem 0 0 0;
 +
display: block;
 +
text-align: center;
 +
width: 100%;
 +
}
 +
 +
ul.actions li:first-child {
 +
padding-top: 0;
 +
}
 +
 +
ul.actions li > * {
 +
width: 100%;
 +
margin: 0 !important;
 +
}
 +
 +
ul.actions li > *.icon:before {
 +
margin-left: -2rem;
 +
}
 +
 +
ul.actions.small li {
 +
padding: 0.5rem 0 0 0;
 +
}
 +
 +
ul.actions.small li:first-child {
 +
padding-top: 0;
 +
}
 +
 +
}
 +
 +
dl {
 +
margin: 0 0 2rem 0;
 +
}
 +
 +
dl dt {
 +
display: block;
 +
font-weight: 700;
 +
margin: 0 0 1rem 0;
 +
}
 +
 +
dl dd {
 +
margin-left: 2rem;
 +
}
 +
 +
ul.alt li {
 +
border-top-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
/* Section/Article */
 +
 +
section.special, article.special {
 +
text-align: center;
 +
}
 +
 +
header p {
 +
position: relative;
 +
margin: 0 0 1.5rem 0;
 +
}
 +
 +
header h2 + p {
 +
font-size: 1.25rem;
 +
margin-top: -1rem;
 +
}
 +
 +
header h3 + p {
 +
font-size: 1.1rem;
 +
margin-top: -0.8rem;
 +
}
 +
 +
header h4 + p,
 +
header h5 + p,
 +
header h6 + p {
 +
font-size: 0.9rem;
 +
margin-top: -0.6rem;
 +
}
 +
 +
header p {
 +
color: #bbb;
 +
}
 +
 +
/* Table */
 +
 +
.table-wrapper {
 +
-webkit-overflow-scrolling: touch;
 +
overflow-x: auto;
 +
}
 +
 +
table {
 +
margin: 0 0 2rem 0;
 +
width: 100%;
 +
}
 +
 +
table tbody tr {
 +
border: solid 1px;
 +
border-left: 0;
 +
border-right: 0;
 +
}
 +
 +
table td {
 +
padding: 0.75rem 0.75rem;
 +
}
 +
 +
table th {
 +
font-size: 0.9rem;
 +
font-weight: 700;
 +
padding: 0 0.75rem 0.75rem 0.75rem;
 +
text-align: left;
 +
}
 +
 +
table thead {
 +
border-bottom: solid 2px;
 +
}
 +
 +
table tfoot {
 +
border-top: solid 2px;
 +
}
 +
 +
table.alt {
 +
border-collapse: separate;
 +
}
 +
 +
table.alt tbody tr td {
 +
border: solid 1px;
 +
border-left-width: 0;
 +
border-top-width: 0;
 +
}
 +
 +
table.alt tbody tr td:first-child {
 +
border-left-width: 1px;
 +
}
 +
 +
table.alt tbody tr:first-child td {
 +
border-top-width: 1px;
 +
}
 +
 +
table.alt thead {
 +
border-bottom: 0;
 +
}
 +
 +
table.alt tfoot {
 +
border-top: 0;
 +
}
 +
 +
table tbody tr {
 +
border-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
table tbody tr:nth-child(2n + 1) {
 +
background-color: rgba(144, 144, 144, 0.075);
 +
}
 +
 +
table th {
 +
color: #555;
 +
}
 +
 +
table thead {
 +
border-bottom-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
table tfoot {
 +
border-top-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
table.alt tbody tr td {
 +
border-color: rgba(144, 144, 144, 0.25);
 +
}
 +
 +
/* Wrapper */
 +
 +
.wrapper {
 +
padding: 6rem 0 4rem 0 ;
 +
position: relative;
 +
margin: 0;
 +
}
 +
 +
.wrapper > .inner {
 +
margin: 0 auto;
 +
width: 80rem;
 +
}
 +
 +
.wrapper.style1 {
 +
background-color: #8a4680;
 +
color: #e2d1df;
 +
}
 +
 +
.wrapper.style1 input, .wrapper.style1 select, .wrapper.style1 textarea {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style1 a {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style1 strong, .wrapper.style1 b {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style1 h1, .wrapper.style1 h2, .wrapper.style1 h3, .wrapper.style1 h4, .wrapper.style1 h5, .wrapper.style1 h6 {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style1 blockquote {
 +
border-left-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 code {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 hr {
 +
border-bottom-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 .box {
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 input[type="submit"],
 +
.wrapper.style1 input[type="reset"],
 +
.wrapper.style1 input[type="button"],
 +
.wrapper.style1 button,
 +
.wrapper.style1 .button {
 +
background-color: #f2f2f2;
 +
color: #000 !important;
 +
}
 +
 +
.wrapper.style1 input[type="submit"]:hover,
 +
.wrapper.style1 input[type="reset"]:hover,
 +
.wrapper.style1 input[type="button"]:hover,
 +
.wrapper.style1 button:hover,
 +
.wrapper.style1 .button:hover {
 +
background-color: white;
 +
}
 +
 +
.wrapper.style1 input[type="submit"]:active,
 +
.wrapper.style1 input[type="reset"]:active,
 +
.wrapper.style1 input[type="button"]:active,
 +
.wrapper.style1 button:active,
 +
.wrapper.style1 .button:active {
 +
background-color: #e5e5e5;
 +
}
 +
 +
.wrapper.style1 input[type="submit"].alt,
 +
.wrapper.style1 input[type="reset"].alt,
 +
.wrapper.style1 input[type="button"].alt,
 +
.wrapper.style1 button.alt,
 +
.wrapper.style1 .button.alt {
 +
background-color: transparent;
 +
box-shadow: inset 0 0 0 2px rgba(255, 255, 255, 0.25);
 +
color: #ffffff !important;
 +
}
 +
 +
.wrapper.style1 input[type="submit"].alt:hover,
 +
.wrapper.style1 input[type="reset"].alt:hover,
 +
.wrapper.style1 input[type="button"].alt:hover,
 +
.wrapper.style1 button.alt:hover,
 +
.wrapper.style1 .button.alt:hover {
 +
background-color: rgba(255, 255, 255, 0.075);
 +
}
 +
 +
.wrapper.style1 input[type="submit"].alt:active,
 +
.wrapper.style1 input[type="reset"].alt:active,
 +
.wrapper.style1 input[type="button"].alt:active,
 +
.wrapper.style1 button.alt:active,
 +
.wrapper.style1 .button.alt:active {
 +
background-color: rgba(255, 255, 255, 0.2);
 +
}
 +
 +
.wrapper.style1 input[type="submit"].alt.icon:before,
 +
.wrapper.style1 input[type="reset"].alt.icon:before,
 +
.wrapper.style1 input[type="button"].alt.icon:before,
 +
.wrapper.style1 button.alt.icon:before,
 +
.wrapper.style1 .button.alt.icon:before {
 +
color: #d0b5cc;
 +
}
 +
 +
.wrapper.style1 input[type="submit"].special,
 +
.wrapper.style1 input[type="reset"].special,
 +
.wrapper.style1 input[type="button"].special,
 +
.wrapper.style1 button.special,
 +
.wrapper.style1 .button.special {
 +
background-color: #ffffff;
 +
color: #8a4680 !important;
 +
}
 +
 +
.wrapper.style1 label {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style1 input[type="text"],
 +
.wrapper.style1 input[type="password"],
 +
.wrapper.style1 input[type="email"],
 +
.wrapper.style1 select,
 +
.wrapper.style1 textarea {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 input[type="text"]:focus,
 +
.wrapper.style1 input[type="password"]:focus,
 +
.wrapper.style1 input[type="email"]:focus,
 +
.wrapper.style1 select:focus,
 +
.wrapper.style1 textarea:focus {
 +
border-color: #ffffff;
 +
box-shadow: 0 0 0 1px #ffffff;
 +
}
 +
 +
.wrapper.style1 .select-wrapper:before {
 +
color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 input[type="checkbox"] + label,
 +
.wrapper.style1 input[type="radio"] + label {
 +
color: #e2d1df;
 +
}
 +
 +
.wrapper.style1 input[type="checkbox"] + label:before,
 +
.wrapper.style1 input[type="radio"] + label:before {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 input[type="checkbox"]:checked + label:before,
 +
.wrapper.style1 input[type="radio"]:checked + label:before {
 +
background-color: #ffffff;
 +
border-color: #ffffff;
 +
color: #8a4680;
 +
}
 +
 +
.wrapper.style1 input[type="checkbox"]:focus + label:before,
 +
.wrapper.style1 input[type="radio"]:focus + label:before {
 +
border-color: #ffffff;
 +
box-shadow: 0 0 0 1px #ffffff;
 +
}
 +
 +
.wrapper.style1 ::-webkit-input-placeholder {
 +
color: #d0b5cc !important;
 +
}
 +
 +
.wrapper.style1 :-moz-placeholder {
 +
color: #d0b5cc !important;
 +
}
 +
 +
.wrapper.style1 ::-moz-placeholder {
 +
color: #d0b5cc !important;
 +
}
 +
 +
.wrapper.style1 :-ms-input-placeholder {
 +
color: #d0b5cc !important;
 +
}
 +
 +
.wrapper.style1 .formerize-placeholder {
 +
color: #d0b5cc !important;
 +
}
 +
 +
.wrapper.style1 ul.alt li {
 +
border-top-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 header p {
 +
color: #d0b5cc;
 +
}
 +
 +
.wrapper.style1 table tbody tr {
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 table tbody tr:nth-child(2n + 1) {
 +
background-color: rgba(255, 255, 255, 0.075);
 +
}
 +
 +
.wrapper.style1 table th {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style1 table thead {
 +
border-bottom-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 table tfoot {
 +
border-top-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style1 table.alt tbody tr td {
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style2 {
 +
background-color: #f2f2f2;
 +
color: #a6a6a6;
 +
}
 +
 +
.wrapper.style2 input, .wrapper.style2 select, .wrapper.style2 textarea {
 +
color: #000;
 +
}
 +
 +
.wrapper.style2 a {
 +
color: #8a4680;
 +
}
 +
 +
.wrapper.style2 strong, .wrapper.style2 b {
 +
color: #000;
 +
}
 +
 +
.wrapper.style2 h1, .wrapper.style2 h2, .wrapper.style2 h3, .wrapper.style2 h4, .wrapper.style2 h5, .wrapper.style2 h6 {
 +
color: #000;
 +
}
 +
 +
.wrapper.style2 blockquote {
 +
border-left-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 code {
 +
background: rgba(0, 0, 0, 0.075);
 +
border-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 hr {
 +
border-bottom-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 .box {
 +
border-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 input[type="submit"],
 +
.wrapper.style2 input[type="reset"],
 +
.wrapper.style2 input[type="button"],
 +
.wrapper.style2 button,
 +
.wrapper.style2 .button {
 +
background-color: #000;
 +
color: #f2f2f2 !important;
 +
}
 +
 +
.wrapper.style2 input[type="submit"].alt,
 +
.wrapper.style2 input[type="reset"].alt,
 +
.wrapper.style2 input[type="button"].alt,
 +
.wrapper.style2 button.alt,
 +
.wrapper.style2 .button.alt {
 +
background-color: transparent;
 +
box-shadow: inset 0 0 0 2px rgba(0, 0, 0, 0.15);
 +
color: #000 !important;
 +
}
 +
 +
.wrapper.style2 input[type="submit"].alt:hover,
 +
.wrapper.style2 input[type="reset"].alt:hover,
 +
.wrapper.style2 input[type="button"].alt:hover,
 +
.wrapper.style2 button.alt:hover,
 +
.wrapper.style2 .button.alt:hover {
 +
background-color: rgba(0, 0, 0, 0.075);
 +
}
 +
 +
.wrapper.style2 input[type="submit"].alt:active,
 +
.wrapper.style2 input[type="reset"].alt:active,
 +
.wrapper.style2 input[type="button"].alt:active,
 +
.wrapper.style2 button.alt:active,
 +
.wrapper.style2 .button.alt:active {
 +
background-color: rgba(0, 0, 0, 0.2);
 +
}
 +
 +
.wrapper.style2 input[type="submit"].alt.icon:before,
 +
.wrapper.style2 input[type="reset"].alt.icon:before,
 +
.wrapper.style2 input[type="button"].alt.icon:before,
 +
.wrapper.style2 button.alt.icon:before,
 +
.wrapper.style2 .button.alt.icon:before {
 +
color: #999999;
 +
}
 +
 +
.wrapper.style2 input[type="submit"].special,
 +
.wrapper.style2 input[type="reset"].special,
 +
.wrapper.style2 input[type="button"].special,
 +
.wrapper.style2 button.special,
 +
.wrapper.style2 .button.special {
 +
background-color: #8a4680;
 +
color: #ffffff !important;
 +
}
 +
 +
.wrapper.style2 input[type="submit"].special:hover,
 +
.wrapper.style2 input[type="reset"].special:hover,
 +
.wrapper.style2 input[type="button"].special:hover,
 +
.wrapper.style2 button.special:hover,
 +
.wrapper.style2 .button.special:hover {
 +
background-color: #9b4f90;
 +
}
 +
 +
.wrapper.style2 input[type="submit"].special:active,
 +
.wrapper.style2 input[type="reset"].special:active,
 +
.wrapper.style2 input[type="button"].special:active,
 +
.wrapper.style2 button.special:active,
 +
.wrapper.style2 .button.special:active {
 +
background-color: #793d70;
 +
}
 +
 +
.wrapper.style2 label {
 +
color: #000;
 +
}
 +
 +
.wrapper.style2 input[type="text"],
 +
.wrapper.style2 input[type="password"],
 +
.wrapper.style2 input[type="email"],
 +
.wrapper.style2 select,
 +
.wrapper.style2 textarea {
 +
background: rgba(0, 0, 0, 0.075);
 +
border-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 input[type="text"]:focus,
 +
.wrapper.style2 input[type="password"]:focus,
 +
.wrapper.style2 input[type="email"]:focus,
 +
.wrapper.style2 select:focus,
 +
.wrapper.style2 textarea:focus {
 +
border-color: #8a4680;
 +
box-shadow: 0 0 0 1px #8a4680;
 +
}
 +
 +
.wrapper.style2 .select-wrapper:before {
 +
color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 input[type="checkbox"] + label,
 +
.wrapper.style2 input[type="radio"] + label {
 +
color: #a6a6a6;
 +
}
 +
 +
.wrapper.style2 input[type="checkbox"] + label:before,
 +
.wrapper.style2 input[type="radio"] + label:before {
 +
background: rgba(0, 0, 0, 0.075);
 +
border-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 input[type="checkbox"]:checked + label:before,
 +
.wrapper.style2 input[type="radio"]:checked + label:before {
 +
background-color: #8a4680;
 +
border-color: #8a4680;
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style2 input[type="checkbox"]:focus + label:before,
 +
.wrapper.style2 input[type="radio"]:focus + label:before {
 +
border-color: #8a4680;
 +
box-shadow: 0 0 0 1px #8a4680;
 +
}
 +
 +
.wrapper.style2 ::-webkit-input-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style2 :-moz-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style2 ::-moz-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style2 :-ms-input-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style2 .formerize-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style2 ul.alt li {
 +
border-top-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 header p {
 +
color: #999999;
 +
}
 +
 +
.wrapper.style2 table tbody tr {
 +
border-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 table tbody tr:nth-child(2n + 1) {
 +
background-color: rgba(0, 0, 0, 0.075);
 +
}
 +
 +
.wrapper.style2 table th {
 +
color: #000;
 +
}
 +
 +
.wrapper.style2 table thead {
 +
border-bottom-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 table tfoot {
 +
border-top-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style2 table.alt tbody tr td {
 +
border-color: rgba(0, 0, 0, 0.15);
 +
}
 +
 +
.wrapper.style3 {
 +
background-color: #000;
 +
color: #bfbfbf;
 +
background-image: url(../../images/bg.jpg);
 +
background-size: cover;
 +
background-attachment: fixed;
 +
background-position: center;
 +
position: relative;
 +
}
 +
 +
.wrapper.style3 input, .wrapper.style3 select, .wrapper.style3 textarea {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style3 a {
 +
color: #8a4680;
 +
}
 +
 +
.wrapper.style3 strong, .wrapper.style3 b {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style3 h1, .wrapper.style3 h2, .wrapper.style3 h3, .wrapper.style3 h4, .wrapper.style3 h5, .wrapper.style3 h6 {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style3 blockquote {
 +
border-left-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 code {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 hr {
 +
border-bottom-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 .box {
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 input[type="submit"],
 +
.wrapper.style3 input[type="reset"],
 +
.wrapper.style3 input[type="button"],
 +
.wrapper.style3 button,
 +
.wrapper.style3 .button {
 +
background-color: #f2f2f2;
 +
color: #000 !important;
 +
}
 +
 +
.wrapper.style3 input[type="submit"]:hover,
 +
.wrapper.style3 input[type="reset"]:hover,
 +
.wrapper.style3 input[type="button"]:hover,
 +
.wrapper.style3 button:hover,
 +
.wrapper.style3 .button:hover {
 +
background-color: white;
 +
}
 +
 +
.wrapper.style3 input[type="submit"]:active,
 +
.wrapper.style3 input[type="reset"]:active,
 +
.wrapper.style3 input[type="button"]:active,
 +
.wrapper.style3 button:active,
 +
.wrapper.style3 .button:active {
 +
background-color: #e5e5e5;
 +
}
 +
 +
.wrapper.style3 input[type="submit"].alt,
 +
.wrapper.style3 input[type="reset"].alt,
 +
.wrapper.style3 input[type="button"].alt,
 +
.wrapper.style3 button.alt,
 +
.wrapper.style3 .button.alt {
 +
background-color: transparent;
 +
box-shadow: inset 0 0 0 2px rgba(255, 255, 255, 0.25);
 +
color: #ffffff !important;
 +
}
 +
 +
.wrapper.style3 input[type="submit"].alt:hover,
 +
.wrapper.style3 input[type="reset"].alt:hover,
 +
.wrapper.style3 input[type="button"].alt:hover,
 +
.wrapper.style3 button.alt:hover,
 +
.wrapper.style3 .button.alt:hover {
 +
background-color: rgba(255, 255, 255, 0.075);
 +
}
 +
 +
.wrapper.style3 input[type="submit"].alt:active,
 +
.wrapper.style3 input[type="reset"].alt:active,
 +
.wrapper.style3 input[type="button"].alt:active,
 +
.wrapper.style3 button.alt:active,
 +
.wrapper.style3 .button.alt:active {
 +
background-color: rgba(255, 255, 255, 0.2);
 +
}
 +
 +
.wrapper.style3 input[type="submit"].alt.icon:before,
 +
.wrapper.style3 input[type="reset"].alt.icon:before,
 +
.wrapper.style3 input[type="button"].alt.icon:before,
 +
.wrapper.style3 button.alt.icon:before,
 +
.wrapper.style3 .button.alt.icon:before {
 +
color: #999999;
 +
}
 +
 +
.wrapper.style3 input[type="submit"].special,
 +
.wrapper.style3 input[type="reset"].special,
 +
.wrapper.style3 input[type="button"].special,
 +
.wrapper.style3 button.special,
 +
.wrapper.style3 .button.special {
 +
background-color: #8a4680;
 +
color: #ffffff !important;
 +
}
 +
 +
.wrapper.style3 input[type="submit"].special:hover,
 +
.wrapper.style3 input[type="reset"].special:hover,
 +
.wrapper.style3 input[type="button"].special:hover,
 +
.wrapper.style3 button.special:hover,
 +
.wrapper.style3 .button.special:hover {
 +
background-color: #9b4f90;
 +
}
 +
 +
.wrapper.style3 input[type="submit"].special:active,
 +
.wrapper.style3 input[type="reset"].special:active,
 +
.wrapper.style3 input[type="button"].special:active,
 +
.wrapper.style3 button.special:active,
 +
.wrapper.style3 .button.special:active {
 +
background-color: #793d70;
 +
}
 +
 +
.wrapper.style3 label {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style3 input[type="text"],
 +
.wrapper.style3 input[type="password"],
 +
.wrapper.style3 input[type="email"],
 +
.wrapper.style3 select,
 +
.wrapper.style3 textarea {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 input[type="text"]:focus,
 +
.wrapper.style3 input[type="password"]:focus,
 +
.wrapper.style3 input[type="email"]:focus,
 +
.wrapper.style3 select:focus,
 +
.wrapper.style3 textarea:focus {
 +
border-color: #8a4680;
 +
box-shadow: 0 0 0 1px #8a4680;
 +
}
 +
 +
.wrapper.style3 .select-wrapper:before {
 +
color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 input[type="checkbox"] + label,
 +
.wrapper.style3 input[type="radio"] + label {
 +
color: #bfbfbf;
 +
}
 +
 +
.wrapper.style3 input[type="checkbox"] + label:before,
 +
.wrapper.style3 input[type="radio"] + label:before {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 input[type="checkbox"]:checked + label:before,
 +
.wrapper.style3 input[type="radio"]:checked + label:before {
 +
background-color: #8a4680;
 +
border-color: #8a4680;
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style3 input[type="checkbox"]:focus + label:before,
 +
.wrapper.style3 input[type="radio"]:focus + label:before {
 +
border-color: #8a4680;
 +
box-shadow: 0 0 0 1px #8a4680;
 +
}
 +
 +
.wrapper.style3 ::-webkit-input-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style3 :-moz-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style3 ::-moz-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style3 :-ms-input-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style3 .formerize-placeholder {
 +
color: #999999 !important;
 +
}
 +
 +
.wrapper.style3 ul.alt li {
 +
border-top-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 header p {
 +
color: #999999;
 +
}
 +
 +
.wrapper.style3 table tbody tr {
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 table tbody tr:nth-child(2n + 1) {
 +
background-color: rgba(255, 255, 255, 0.075);
 +
}
 +
 +
.wrapper.style3 table th {
 +
color: #ffffff;
 +
}
 +
 +
.wrapper.style3 table thead {
 +
border-bottom-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 table tfoot {
 +
border-top-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 table.alt tbody tr td {
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.wrapper.style3 .inner {
 +
position: relative;
 +
z-index: 2;
 +
}
 +
 +
.wrapper.style3 p {
 +
text-transform: uppercase;
 +
font-size: .75rem;
 +
font-weight: 300;
 +
margin: 0 0 .5rem 0;
 +
padding: 0 0 1rem 0;
 +
letter-spacing: .25rem;
 +
}
 +
 +
.wrapper.style3 p:after {
 +
content: '';
 +
position: absolute;
 +
margin: auto;
 +
right: 0;
 +
bottom: 0;
 +
left: 0;
 +
width: 50%;
 +
height: 1px;
 +
background-color: rgba(255, 255, 255, 0.5);
 +
}
 +
 +
.wrapper.style3:before {
 +
-moz-transition: opacity 3s ease;
 +
-webkit-transition: opacity 3s ease;
 +
-ms-transition: opacity 3s ease;
 +
transition: opacity 3s ease;
 +
-moz-transition-delay: 1.25s;
 +
-webkit-transition-delay: 1.25s;
 +
-ms-transition-delay: 1.25s;
 +
transition-delay: 1.25s;
 +
content: '';
 +
display: block;
 +
background-color: rgba(0, 0, 0, 0.95);
 +
height: 100%;
 +
left: 0;
 +
opacity: 0.75;
 +
position: absolute;
 +
top: 0;
 +
width: 100%;
 +
z-index: 1;
 +
}
 +
 +
@media screen and (max-width: 1280px) {
 +
 +
.wrapper > .inner {
 +
width: 75rem;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
.wrapper > .inner {
 +
width: 90%;
 +
}
 +
 +
.wrapper.style3 {
 +
background-attachment: scroll;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
.wrapper {
 +
padding: 3rem 0 1rem 0 ;
 +
}
 +
 +
}
 +
 +
/* Flexgrid */
 +
 +
.grid-style {
 +
width: 100%;
 +
margin: 0 0 2.5rem 0;
 +
display: -moz-flex;
 +
display: -webkit-flex;
 +
display: -ms-flex;
 +
display: flex;
 +
-moz-flex-wrap: wrap;
 +
-webkit-flex-wrap: wrap;
 +
-ms-flex-wrap: wrap;
 +
flex-wrap: wrap;
 +
-moz-align-items: stretch;
 +
-webkit-align-items: stretch;
 +
-ms-align-items: stretch;
 +
align-items: stretch;
 +
}
 +
 +
.grid-style > * {
 +
-moz-flex-shrink: 1;
 +
-webkit-flex-shrink: 1;
 +
-ms-flex-shrink: 1;
 +
flex-shrink: 1;
 +
-moz-flex-grow: 0;
 +
-webkit-flex-grow: 0;
 +
-ms-flex-grow: 0;
 +
flex-grow: 0;
 +
}
 +
 +
.grid-style > * {
 +
width: 50%;
 +
}
 +
 +
.grid-style > * {
 +
padding: 2rem;
 +
width: calc(50% + 2rem);
 +
}
 +
 +
.grid-style > :nth-child(-n + 2) {
 +
padding-top: 0;
 +
}
 +
 +
.grid-style > :nth-last-child(-n + 2) {
 +
padding-bottom: 0;
 +
}
 +
 +
.grid-style > :nth-child(2n + 1) {
 +
padding-left: 0;
 +
}
 +
 +
.grid-style > :nth-child(2n) {
 +
padding-right: 0;
 +
}
 +
 +
.grid-style > :nth-child(2n + 1),
 +
.grid-style > :nth-child(2n) {
 +
width: calc(50% + 0rem);
 +
}
 +
 +
.grid-style .box {
 +
margin: 0;
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
.grid-style > * {
 +
width: 100%;
 +
}
 +
 +
.grid-style > * {
 +
padding: 1rem;
 +
width: calc(50% + 1rem);
 +
}
 +
 +
.grid-style > :nth-child(-n + 2) {
 +
padding-top: 1rem;
 +
}
 +
 +
.grid-style > :nth-last-child(-n + 2) {
 +
padding-bottom: 1rem;
 +
}
 +
 +
.grid-style > :nth-child(2n + 1) {
 +
padding-left: 1rem;
 +
}
 +
 +
.grid-style > :nth-child(2n) {
 +
padding-right: 1rem;
 +
}
 +
 +
.grid-style > :nth-child(2n + 1),
 +
.grid-style > :nth-child(2n) {
 +
padding: 1rem;
 +
width: calc(100% + 2rem);
 +
}
 +
 +
.grid-style > * {
 +
padding: 1rem;
 +
width: calc(100% + 2rem);
 +
}
 +
 +
.grid-style > :nth-child(-n + 1) {
 +
padding-top: 0;
 +
}
 +
 +
.grid-style > :nth-last-child(-n + 1) {
 +
padding-bottom: 0;
 +
}
 +
 +
.grid-style > :nth-child(1n + 1) {
 +
padding-left: 0;
 +
}
 +
 +
.grid-style > :nth-child(1n) {
 +
padding-right: 0;
 +
}
 +
 +
.grid-style > :nth-child(1n + 1),
 +
.grid-style > :nth-child(1n) {
 +
width: calc(100% + 1rem);
 +
}
 +
 +
}
 +
 +
.gallery {
 +
width: 100%;
 +
margin: 2.5rem 0 2.5rem 0;
 +
display: -moz-flex;
 +
display: -webkit-flex;
 +
display: -ms-flex;
 +
display: flex;
 +
-moz-flex-wrap: wrap;
 +
-webkit-flex-wrap: wrap;
 +
-ms-flex-wrap: wrap;
 +
flex-wrap: wrap;
 +
-moz-align-items: stretch;
 +
-webkit-align-items: stretch;
 +
-ms-align-items: stretch;
 +
align-items: stretch;
 +
}
 +
 +
.gallery > * {
 +
-moz-flex-shrink: 1;
 +
-webkit-flex-shrink: 1;
 +
-ms-flex-shrink: 1;
 +
flex-shrink: 1;
 +
-moz-flex-grow: 0;
 +
-webkit-flex-grow: 0;
 +
-ms-flex-grow: 0;
 +
flex-grow: 0;
 +
}
 +
 +
.gallery > * {
 +
width: 50%;
 +
}
 +
 +
.gallery > * {
 +
padding: 2rem;
 +
width: calc(50% + 2rem);
 +
}
 +
 +
.gallery > :nth-child(-n + 2) {
 +
padding-top: 0;
 +
}
 +
 +
.gallery > :nth-last-child(-n + 2) {
 +
padding-bottom: 0;
 +
}
 +
 +
.gallery > :nth-child(2n + 1) {
 +
padding-left: 0;
 +
}
 +
 +
.gallery > :nth-child(2n) {
 +
padding-right: 0;
 +
}
 +
 +
.gallery > :nth-child(2n + 1),
 +
.gallery > :nth-child(2n) {
 +
width: calc(50% + 0rem);
 +
}
 +
 +
.gallery .image {
 +
background: #FFF;
 +
padding: 1rem;
 +
}
 +
 +
.gallery .image.fit {
 +
margin: 0;
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
.gallery > * {
 +
width: 100%;
 +
}
 +
 +
.gallery > * {
 +
padding: 1rem;
 +
width: calc(50% + 1rem);
 +
}
 +
 +
.gallery > :nth-child(-n + 2) {
 +
padding-top: 1rem;
 +
}
 +
 +
.gallery > :nth-last-child(-n + 2) {
 +
padding-bottom: 1rem;
 +
}
 +
 +
.gallery > :nth-child(2n + 1) {
 +
padding-left: 1rem;
 +
}
 +
 +
.gallery > :nth-child(2n) {
 +
padding-right: 1rem;
 +
}
 +
 +
.gallery > :nth-child(2n + 1),
 +
.gallery > :nth-child(2n) {
 +
padding: 1rem;
 +
width: calc(100% + 2rem);
 +
}
 +
 +
.gallery > * {
 +
padding: 1rem;
 +
width: calc(100% + 2rem);
 +
}
 +
 +
.gallery > :nth-child(-n + 1) {
 +
padding-top: 0;
 +
}
 +
 +
.gallery > :nth-last-child(-n + 1) {
 +
padding-bottom: 0;
 +
}
 +
 +
.gallery > :nth-child(1n + 1) {
 +
padding-left: 0;
 +
}
 +
 +
.gallery > :nth-child(1n) {
 +
padding-right: 0;
 +
}
 +
 +
.gallery > :nth-child(1n + 1),
 +
.gallery > :nth-child(1n) {
 +
width: calc(100% + 1rem);
 +
}
 +
 +
}
 +
 +
/* Header */
 +
 +
body.subpage {
 +
padding-top: 3.25em;
 +
}
 +
 +
@-moz-keyframes reveal-header {
 +
0% {
 +
top: -4em;
 +
opacity: 0;
 +
}
 +
 +
100% {
 +
top: 0;
 +
opacity: 1;
 +
}
 +
}
 +
 +
@-webkit-keyframes reveal-header {
 +
0% {
 +
top: -4em;
 +
opacity: 0;
 +
}
 +
 +
100% {
 +
top: 0;
 +
opacity: 1;
 +
}
 +
}
 +
 +
@-ms-keyframes reveal-header {
 +
0% {
 +
top: -4em;
 +
opacity: 0;
 +
}
 +
 +
100% {
 +
top: 0;
 +
opacity: 1;
 +
}
 +
}
 +
 +
@keyframes reveal-header {
 +
0% {
 +
top: -4em;
 +
opacity: 0;
 +
}
 +
 +
100% {
 +
top: 0;
 +
opacity: 1;
 +
}
 +
}
 +
 +
#header {
 +
background: rgba(0, 0, 0, 0.975);
 +
color: #a6a6a6;
 +
cursor: default;
 +
height: 3.25em;
 +
left: 0;
 +
line-height: 3.25em;
 +
position: fixed;
 +
text-align: right;
 +
top: 0;
 +
width: 100%;
 +
z-index: 10001;
 +
}
 +
 +
#header > .logo {
 +
display: inline-block;
 +
height: inherit;
 +
left: 1.25em;
 +
line-height: inherit;
 +
margin: 0;
 +
padding: 0;
 +
position: absolute;
 +
top: 0;
 +
}
 +
 +
#header > .logo a {
 +
font-size: 1.25em;
 +
color: #FFF;
 +
text-decoration: none;
 +
}
 +
 +
#header > .logo a:hover {
 +
color: rgba(255, 255, 255, 0.65);
 +
}
 +
 +
#header > .logo span {
 +
font-weight: 400;
 +
font-size: .8em;
 +
color: rgba(255, 255, 255, 0.65);
 +
}
 +
 +
#header > a {
 +
-moz-transition: color 0.2s ease-in-out;
 +
-webkit-transition: color 0.2s ease-in-out;
 +
-ms-transition: color 0.2s ease-in-out;
 +
transition: color 0.2s ease-in-out;
 +
display: inline-block;
 +
padding: 0 0.75em;
 +
color: inherit;
 +
text-decoration: none;
 +
color: #FFF;
 +
}
 +
 +
#header > a:hover {
 +
color: #f2f2f2;
 +
}
 +
 +
#header > a[href="#menu"] {
 +
text-decoration: none;
 +
-webkit-tap-highlight-color: transparent;
 +
}
 +
 +
#header > a[href="#menu"]:before {
 +
content: "";
 +
-moz-osx-font-smoothing: grayscale;
 +
-webkit-font-smoothing: antialiased;
 +
font-family: FontAwesome;
 +
font-style: normal;
 +
font-weight: normal;
 +
text-transform: none !important;
 +
}
 +
 +
#header > a[href="#menu"]:before {
 +
margin: 0 0.5em 0 0;
 +
}
 +
 +
#header > a + a[href="#menu"]:last-child {
 +
border-left: solid 1px rgba(0, 0, 0, 0.15);
 +
padding-left: 1.25em;
 +
margin-left: 0.5em;
 +
}
 +
 +
#header > a:last-child {
 +
padding-right: 1.25em;
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
#header > a {
 +
padding: 0 0.5em;
 +
}
 +
 +
#header > a + a[href="#menu"]:last-child {
 +
padding-left: 1em;
 +
margin-left: 0.25em;
 +
}
 +
 +
#header > a:last-child {
 +
padding-right: 1em;
 +
}
 +
 +
}
 +
 +
#header.reveal {
 +
-moz-animation: reveal-header 0.5s ease;
 +
-webkit-animation: reveal-header 0.5s ease;
 +
-ms-animation: reveal-header 0.5s ease;
 +
animation: reveal-header 0.5s ease;
 +
}
 +
 +
#header.alt {
 +
-moz-animation: none;
 +
-webkit-animation: none;
 +
-ms-animation: none;
 +
animation: none;
 +
background-color: transparent;
 +
box-shadow: none;
 +
overflow: hidden;
 +
position: absolute;
 +
top: 1.5em;
 +
}
 +
 +
#header.alt h1 {
 +
left: 2.5em;
 +
}
 +
 +
#header.alt nav {
 +
right: 2.5em;
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
body.subpage {
 +
padding-top: 44px;
 +
}
 +
 +
#header {
 +
height: 44px;
 +
line-height: 44px;
 +
}
 +
 +
#header > h1 {
 +
left: 1em;
 +
}
 +
 +
#header > h1 a {
 +
font-size: 1em;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 480px) {
 +
 +
#header {
 +
min-width: 320px;
 +
}
 +
 +
}
 +
 +
/* Menu */
 +
 +
#menu {
 +
-moz-transform: translateX(20rem);
 +
-webkit-transform: translateX(20rem);
 +
-ms-transform: translateX(20rem);
 +
transform: translateX(20rem);
 +
-moz-transition: -moz-transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
-webkit-transition: -webkit-transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
-ms-transition: -ms-transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
transition: transform 0.5s ease, box-shadow 0.5s ease, visibility 0.5s;
 +
-webkit-overflow-scrolling: touch;
 +
background: rgba(0, 0, 0, 0.95);
 +
box-shadow: none;
 +
color: #000;
 +
height: 100%;
 +
max-width: 80%;
 +
overflow-y: auto;
 +
padding: 3rem 2rem;
 +
position: fixed;
 +
right: 0;
 +
top: 0;
 +
visibility: hidden;
 +
width: 20rem;
 +
z-index: 10002;
 +
}
 +
 +
#menu > ul {
 +
margin: 0 0 1rem 0;
 +
}
 +
 +
#menu > ul.links {
 +
list-style: none;
 +
padding: 0;
 +
}
 +
 +
#menu > ul.links > li {
 +
padding: 0;
 +
}
 +
 +
#menu > ul.links > li > a:not(.button) {
 +
border: 0;
 +
border-top: solid 1px rgba(255, 255, 255, 0.125);
 +
color: rgba(255, 255, 255, 0.5);
 +
display: block;
 +
line-height: 3.5rem;
 +
text-decoration: none;
 +
text-transform: uppercase;
 +
}
 +
 +
#menu > ul.links > li > a:not(.button):hover {
 +
color: #FFF;
 +
}
 +
 +
#menu > ul.links > li > .button {
 +
display: block;
 +
margin: 0.5rem 0 0 0;
 +
}
 +
 +
#menu > ul.links > li:first-child > a:not(.button) {
 +
border-top: 0 !important;
 +
}
 +
 +
#menu .close {
 +
text-decoration: none;
 +
-moz-transition: color 0.2s ease-in-out;
 +
-webkit-transition: color 0.2s ease-in-out;
 +
-ms-transition: color 0.2s ease-in-out;
 +
transition: color 0.2s ease-in-out;
 +
-webkit-tap-highlight-color: transparent;
 +
border: 0;
 +
color: #999999;
 +
cursor: pointer;
 +
display: block;
 +
height: 3.25rem;
 +
line-height: 3.25rem;
 +
padding-right: 1.25rem;
 +
position: absolute;
 +
right: 0;
 +
text-align: right;
 +
top: 0;
 +
vertical-align: middle;
 +
width: 7rem;
 +
}
 +
 +
#menu .close:before {
 +
-moz-osx-font-smoothing: grayscale;
 +
-webkit-font-smoothing: antialiased;
 +
font-family: FontAwesome;
 +
font-style: normal;
 +
font-weight: normal;
 +
text-transform: none !important;
 +
}
 +
 +
#menu .close:before {
 +
content: '\f00d';
 +
font-size: 1.25rem;
 +
}
 +
 +
#menu .close:hover {
 +
color: #000;
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
#menu .close {
 +
height: 4rem;
 +
line-height: 4rem;
 +
}
 +
 +
}
 +
 +
#menu.visible {
 +
-moz-transform: translateX(0);
 +
-webkit-transform: translateX(0);
 +
-ms-transform: translateX(0);
 +
transform: translateX(0);
 +
box-shadow: 0 0 1.5rem 0 rgba(0, 0, 0, 0.2);
 +
visibility: visible;
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
#menu {
 +
padding: 2.5rem 1.75rem;
 +
}
 +
 +
}
 +
 +
/* Banner */
 +
 +
.banner {
 +
background-color: #8a4680;
 +
color: #e2d1df;
 +
padding: 8em 0;
 +
position: relative;
 +
}
 +
 +
.banner input, .banner select, .banner textarea {
 +
color: #ffffff;
 +
}
 +
 +
.banner a {
 +
color: #ffffff;
 +
}
 +
 +
.banner strong, .banner b {
 +
color: #ffffff;
 +
}
 +
 +
.banner h1, .banner h2, .banner h3, .banner h4, .banner h5, .banner h6 {
 +
color: #ffffff;
 +
}
 +
 +
.banner blockquote {
 +
border-left-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.banner code {
 +
background: rgba(255, 255, 255, 0.075);
 +
border-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.banner hr {
 +
border-bottom-color: rgba(255, 255, 255, 0.25);
 +
}
 +
 +
.banner.full {
 +
padding: 0;
 +
min-height: 100vh;
 +
height: 100vh !important;
 +
}
 +
 +
.banner.half {
 +
padding: 0;
 +
min-height: 50vh;
 +
height: 50vh !important;
 +
}
 +
 +
.banner:after {
 +
-moz-pointer-events: none;
 +
-webkit-pointer-events: none;
 +
-ms-pointer-events: none;
 +
pointer-events: none;
 +
-moz-transition: opacity 1.5s ease-in-out, visibility 1.5s;
 +
-webkit-transition: opacity 1.5s ease-in-out, visibility 1.5s;
 +
-ms-transition: opacity 1.5s ease-in-out, visibility 1.5s;
 +
transition: opacity 1.5s ease-in-out, visibility 1.5s;
 +
background: #000000;
 +
content: '';
 +
display: block;
 +
height: 100%;
 +
left: 0;
 +
opacity: 0;
 +
position: absolute;
 +
top: 0;
 +
visibility: hidden;
 +
width: 100%;
 +
z-index: 2;
 +
}
 +
 +
.banner .indicators {
 +
bottom: 1.5em;
 +
left: 0;
 +
list-style: none;
 +
margin: 0;
 +
padding: 0;
 +
position: absolute;
 +
text-align: center;
 +
width: 100%;
 +
z-index: 2;
 +
}
 +
 +
.banner .indicators li {
 +
cursor: pointer;
 +
display: inline-block;
 +
height: 2em;
 +
overflow: hidden;
 +
padding: 0;
 +
position: relative;
 +
text-indent: 2em;
 +
width: 2em;
 +
}
 +
 +
.banner .indicators li:before {
 +
background: rgba(255, 255, 255, 0.35);
 +
border-radius: 100%;
 +
content: '';
 +
display: inline-block;
 +
height: 0.8em;
 +
left: 50%;
 +
margin: -0.4em 0 0 -0.4em;
 +
position: absolute;
 +
text-indent: 0;
 +
top: 50%;
 +
width: 0.8em;
 +
}
 +
 +
.banner .indicators li.visible:before {
 +
background: #fff;
 +
}
 +
 +
.banner > article {
 +
-moz-transition: opacity 1.5s ease, visibility 1.5s;
 +
-webkit-transition: opacity 1.5s ease, visibility 1.5s;
 +
-ms-transition: opacity 1.5s ease, visibility 1.5s;
 +
transition: opacity 1.5s ease, visibility 1.5s;
 +
background-attachment: fixed;
 +
background-position: center;
 +
background-repeat: no-repeat;
 +
background-size: cover;
 +
height: 100%;
 +
left: 0;
 +
opacity: 0;
 +
position: absolute;
 +
text-align: center;
 +
top: 0;
 +
visibility: hidden;
 +
width: 100%;
 +
z-index: 0;
 +
}
 +
 +
.banner > article:before {
 +
content: '';
 +
display: inline-block;
 +
height: 100%;
 +
vertical-align: middle;
 +
}
 +
 +
.banner > article:after {
 +
content: '';
 +
display: block;
 +
width: 100%;
 +
height: 100%;
 +
position: absolute;
 +
top: 0;
 +
left: 0;
 +
background: #000;
 +
opacity: 0.35;
 +
}
 +
 +
.banner > article .inner {
 +
position: relative;
 +
display: inline-block;
 +
vertical-align: middle;
 +
z-index: 1;
 +
}
 +
 +
.banner > article .inner > :last-child {
 +
margin-bottom: 0;
 +
}
 +
 +
.banner > article h2 {
 +
font-size: 7rem;
 +
margin-bottom: 0;
 +
color: #FFF;
 +
font-weight: 300;
 +
}
 +
 +
.banner > article h2:after {
 +
display: none;
 +
}
 +
 +
.banner > article p {
 +
color: rgba(255, 255, 255, 0.65);
 +
text-transform: uppercase;
 +
font-size: 1rem;
 +
font-weight: 300;
 +
margin: 0;
 +
padding-bottom: 1.75rem;
 +
letter-spacing: .25rem;
 +
}
 +
 +
.banner > article p:after {
 +
content: '';
 +
position: absolute;
 +
margin: auto;
 +
right: 0;
 +
bottom: 0;
 +
left: 0;
 +
width: 50%;
 +
height: 1px;
 +
background-color: rgba(255, 255, 255, 0.65);
 +
}
 +
 +
.banner > article a {
 +
color: #FFF;
 +
text-decoration: none;
 +
}
 +
 +
.banner > article img {
 +
display: none;
 +
}
 +
 +
.banner > article.visible {
 +
opacity: 1;
 +
visibility: visible;
 +
}
 +
 +
.banner > article.top {
 +
z-index: 1;
 +
}
 +
 +
.banner > article.instant {
 +
-moz-transition: none !important;
 +
-webkit-transition: none !important;
 +
-ms-transition: none !important;
 +
transition: none !important;
 +
}
 +
 +
body.is-loading .banner:after {
 +
opacity: 1.0;
 +
visibility: visible;
 +
}
 +
 +
@media screen and (max-width: 1280px) {
 +
 +
.banner.full {
 +
padding: 0;
 +
min-height: 75vh;
 +
height: 75vh !important;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 980px) {
 +
 +
.banner.full {
 +
padding: 0;
 +
min-height: 50vh;
 +
height: 50vh !important;
 +
}
 +
 +
.banner > article {
 +
background-attachment: scroll;
 +
}
 +
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
.banner > article .inner {
 +
width: 90%;
 +
}
 +
 +
.banner > article p {
 +
margin-bottom: 1rem;
 +
}
 +
 +
.banner > article h2 {
 +
font-size: 4em;
 +
}
 +
 +
}
 +
 +
body.is-mobile .banner > article {
 +
background-attachment: scroll;
 +
}
 +
 +
/* Main */
 +
 +
#main {
 +
padding: 4rem 0 2rem 0 ;
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
#main {
 +
padding: 3rem 0 1rem 0 ;
 +
}
 +
 +
}
 +
 +
/* Footer */
 +
 +
#footer {
 +
padding: 2rem 0 2rem 0 ;
 +
background: #000;
 +
text-align: center;
 +
}
 +
 +
#footer a {
 +
color: rgba(255, 255, 255, 0.5);
 +
}
 +
 +
#footer a:hover {
 +
color: #FFF;
 +
}
 +
 +
#footer .copyright {
 +
color: #bbb;
 +
font-size: 0.9rem;
 +
margin: 0 0 2rem 0;
 +
padding: 0;
 +
text-align: center;
 +
}
 +
 +
@media screen and (max-width: 736px) {
 +
 +
#footer {
 +
padding: 3rem 0 1rem 0 ;
 +
}
 +
 +
}
 +
</style>
 +
</head>
 +
<body>
 +
 +
<!-- Header -->
 +
<!-- Navbar -->
 +
<div class="w3-top" style="z-index: 4">
 +
  <div class="w3-bar w3-black w3-card-2 w3-left-align w3-large">
 +
    <a class="w3-bar-item w3-button w3-hide-medium w3-hide-large w3-right w3-padding-large w3-hover-white w3-large w3-red" href="javascript:void(0);" onclick="myFunction()" title="Toggle Navigation Menu"><i class="fa fa-bars"></i></a>
 +
    <a href="https://2017.igem.org/Team:Mingdao" class="w3-bar-item w3-button w3-padding-large w3-white">Home</a>
 +
    <a href="#" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-blue w3-hover-white" style="text-decoration: none; color: white">Achievements</a>
 +
    <a href="https://2017.igem.org/Team:Mingdao/Demonstrate" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white">Project</a>
 +
    <a href="https://2017.igem.org/Team:Mingdao/HP/Silver" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white">Human-Practice</a>
 +
    <a href="#" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white">Notebook</a>
 +
    <a href="https://2017.igem.org/Team:Mingdao/Attributions" class="w3-bar-item w3-button w3-hide-small w3-padding-large w3-hover-white" style="text-decoration: none; color: white;">Attributions</a>
 +
  </div>
 +
</div>
 +
        <!-- Navbar END -->
 +
 +
 +
<!-- Sidebar/menu -->
 +
<nav class="w3-sidebar w3-collapse w3-animate-left" style="z-index:3;width:180px; background-color: #F8F9FA" id="mySidebar"><br>
 +
 +
    <div class="w3-container">
 +
        <h5>Dashboard</h5>
 +
    </div>
 +
 +
            <div class="w3-bar-block">
 +
                <form action="https://2017.igem.org/Team:Mingdao/Model" style="margin: 10px 15px -10px 0;">
 +
                    <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem;"> <i class="fa fa-bullseye fa-fw"></i>  Model</button>
 +
                </form>
 +
                <form action="https://2017.igem.org/Team:Mingdao/Part_Collection" style="margin: 0 15px -10px 0;">
 +
                    <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem"> <i class="fa fa-bullseye fa-fw"></i> Parts</button>
 +
                </form>
 +
                <form action="https://2017.igem.org/Team:Mingdao/Safety" style="margin: 0 15px -10px 0;">
 +
                    <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem"> <i class="fa fa-bullseye fa-fw"></i> Safety</button>
 +
                </form>
 +
                <form action="https://2017.igem.org/Team:Mingdao/InterLab" style="margin: 0 15px -20px 0;">
 +
                    <button type="submit" class="btn btn-primary btn-block text-white" style="text-align: left; padding-left:1rem;font-size: 1.2rem"> <i class="fa fa-bullseye fa-fw"></i> Interlab</button>
 +
                </form>
 +
                <hr>
 +
            </div>
 +
 +
  <div class="w3-bar-block">
 +
    <a href="#" class="w3-bar-item w3-button w3-padding-16 w3-hide-large w3-dark-grey w3-hover-black" onclick="w3_close()" title="close menu"><i class="fa fa-remove fa-fw"></i>  Close Menu</a>
 +
  </div>
 +
    <nav class="navbar navbar-light bg-light">
 +
      <a class="navbar-brand btn" href="#" style="text-decoration: none;"><b style="letter-spacing: 0.2rem">Overview</b></a>
 +
      <a class="navbar-brand btn" href="#recomb-md" style="text-decoration: none">Recombination</a>
 +
      <a class="navbar-brand btn" href="#suicide-md" style="text-decoration: none">Suicide</a>
 +
      <a class="navbar-brand btn" href="#transport-md" style="text-decoration: none">Transporter</a>
 +
    </nav>
 +
</nav>
 +
<!-- Overlay effect when opening sidebar on small screens -->
 +
<div class="w3-overlay w3-hide-large w3-animate-opacity" onclick="w3_close()" style="cursor:pointer" title="close side menu" id="myOverlay"></div>
 +
 +
<div style="margin-left: 180px; background-color: #f2f2f2">
 +
        <!-- Jumbotron -->
 +
        <div class="jumbotron" style="margin-top: 1rem; background: none; background-color: #fff">
 +
          <img src="https://static.igem.org/mediawiki/2017/2/2a/Mingdaojay1003-1.jpeg" width="60%" style="display: block; margin: auto">
 +
        </div>
 +
<!-- One -->
 +
        <section id="intro" style="margin-bottom: 10rem">
 +
 +
            <!-- MINGDAO -->
 +
            <div class="row">
 +
                <div class="col-md-8 col-sm-10" style="display: block; margin: auto">
 +
                        <div class="card">
 +
                          <h4 class="card-header text-center" style="letter-spacing: 0.2rem; font-size:1.5rem; background-color: #004480; color: white">Overview</h4>
 +
                          <div class="card-body">
 +
                            <div class="row">
 +
                                <div class="col-md-12 col-sm-12">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/1/15/Mingdaojay1003-3.jpeg" width="100%">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/9/9c/Mingdaojay1003-4.jpeg" width="100%">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/f9/Mingdaojay1003-5.jpeg" width="100%">
 +
                                </div>
 +
                            </div>
 +
 +
                          </div>
 +
                        </div>
 +
                </div>
 +
            </div>
 +
            <!-- MINGDAO END -->
 +
            <!-- Recombination start -->
 +
            <div id="recomb-md">
 +
                <div class="row w3-padding-32">
 +
                    <div class="col-md-2 col-sm-3" style="background-color: #fff"></div>
 +
                    <div class="col-md-8 col-sm-3" style="background-color: #fff">
 +
                        <img src="https://static.igem.org/mediawiki/2017/c/c4/Mingdaojay1003-6.jpeg" width="100%">
 +
                    </div>
 +
                    <div class="col-md-2 col-sm-3" style="background-color: #fff"></div>
 +
                </div>
 +
            </div>
 +
            <div class="row">
 +
                <div class="col-md-10 col-sm-12" style="display: block; margin: auto">
 +
 +
                <div class="card">
 +
                      <div class="card-header text-center" style="color: white; letter-spacing: 0.3rem;font-size:1.8rem; background-color:#004480">
 +
                        Background
 +
                      </div>
 +
                      <div class="card-body" style="background-image: url(https://static.igem.org/mediawiki/2017/6/65/MD-test-floater-bg-image.png); background-size: 100%;">
 +
 +
                        <h4 class="card-title w3-padding-24">
 +
                            To engineer <i>Lactobacillus acidophilus</i> by chromosome integration through homologous recombination, the selection of integration site is very important for successful recombination and not disturbing bacterial internal metabolism.
 +
                        </h4>
 +
                        <h4 class="card-title w3-padding-24">
 +
                            Based on the method created by Grace L. Douglas and Todd R. Klaenhammer<sup>1</sup>, the region between <i>slpA</i> gene (LBA0169) stop codon and the terminator was chosen as the intergenic insertion location.  The gene of <i>slpA</i> encodes a surface-layer protein with a strong constitutive promoter activity, which can also drive the expression of the inserted gene.
 +
                        </h4>
 +
 +
                        <div class="row">
 +
                            <div class="col-md-10 col-sm-12" style="display: block; margin: auto">
 +
                                <img src="https://static.igem.org/mediawiki/2017/1/1e/Mingdaochuck1017-16.png" width="100%" style="display: block; margin: auto;">
 +
                                <h4 style="padding-left: 3rem"><b>Fig 1. The schematic diagram of gene integration location (modified from Appl Environ Microbiol. 2011)</b></h4>
 +
                            </div>
 +
                        </div>
 +
 +
                        <button class="btn text-white" style="letter-spacing: 0.3rem;background-color: #004480">
 +
                        <span class="badge badge-info">1</span>
 +
                          &nbsp;&nbsp;&nbsp;Recombination Vector – pLBA169
 +
                          <span class="sr-only"></span>
 +
                        </button>
 +
                        <h4 class="w3-padding-16">
 +
                            To make pLBA169, the downstream region of LBA0169 and the upstream region of the terminator were amplified by PCR. Then, the two PCR-amplified DNA fragments were ligated to pSB1C3 by tripartite ligation.
 +
                        </h4>
 +
                        <h4 class="w3-padding-16">
 +
                            Next, CP29-RBS-aeBlue was cut from Part: BBa_K1033280. And this gel isolated DNA fragment was further inserted between two recombination regions on pSB1C3. This process created the standard EcoRI-XbaI-PART-SpeI-PstI assembly position.
 +
                        </h4>
 +
                        <h4 class="w3-padding-16">
 +
                            To delete the extra SpeI recognition site within the one of the recombination region, we performed site-directed mutagenesis to change one nucleotide of SpeI recognition sequence. The final product has been confirmed by DNA sequencing.
 +
                        </h4>
 +
                        <h4 class="w3-padding-16">
 +
                            You can refer to the following flow chart of gene cloning of the recombination vector.
 +
                        </h4>
 +
                        <div class="col-md-12 col-sm-12" style="margin-top: 5rem">
 +
                            <img src="https://static.igem.org/mediawiki/2017/5/5b/Mingdaochuck1017-17.png" width="80%" style="display: block; margin: auto">
 +
                        </div>
 +
                        <!-- REFERNCE -->
 +
                        <div class="col-md-12 col-sm-12" >
 +
                            <div style="margin-left: 20px; margin-top: 3rem">
 +
                                <!-- Box -->
 +
                                <div style="margin-right: 50px">
 +
                                    <ul class="list-group">
 +
                                      <li class="list-group-item w3-wide" style="text-align: center"><strong>- REFERENCE -</strong></li>
 +
                                      <li class="list-group-item" style="color: black;">1. &nbsp;&nbsp;&nbsp;&nbsp;Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM. Appl Environ Microbiol. 2011;77(20):7365-71.</li>
 +
 +
                                    </ul>
 +
                                </div>
 +
                            </div>
 +
                            <!-- REFERNCE END -->
 +
                          </div>
 +
                        </div>
 +
                    </div>
 +
                    <!-- New card -->
 +
                        <div class="card" style="width: 100%; margin-top: 3rem; margin-bottom: 3rem">
 +
                            <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/c/c1/Mingdaojay1003-7.jpeg" alt="Card image cap">
 +
                          <div class="card-body">
 +
                            <!-- PART start -->
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230000</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Type:  </b>Composite part (device)</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                Firstly, the elements of upstream and downstream recombination sites were replicated from gDNA of Lactobacillus acidophilus and put onto pSB1C3 as a L. acidophilus recombination vector, named pLBA169, at EcoRI and PstI locations. Secondly, to generate as a standard BioBrick part and assembly vector, the part of Promoter CP29-RBS-aeBlue (BBa_K1033280) was cut by EcoRI and PstI and assembled with the vector. Finally, because there’s a SpeI recognition sequence within the 169dn element, site-directed mutagenesis was performed to change a nucleotide to leave a single SpeI site in the BioBrick suffix region. (See the flow chart and data below).
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                A vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA (LBA0169), which encodes a surface layer protein A. The aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will express blue color proteins.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <button class="btn text-white" style="letter-spacing: 0.3rem;background-color: #004480; display: block; margin: auto; margin-bottom: 3rem; margin-top: 3rem">
 +
                                <span style="padding: 0 8rem 0 8rem">Gene cloning for LBA169</span>
 +
                            </button>
 +
                            <!-- Card Group -->
 +
                            <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                <!-- first -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/5/5f/Mingdaojay1003-15.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- second -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/d/d3/Mingdaojay1003-16.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                            </div>
 +
                            <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                <!-- third -->
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/e/ef/Mingdaojay1003-17.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth -->
 +
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/1/15/Mingdaojay1003-18.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth END -->
 +
                            </div>
 +
                            <!-- fifth -->
 +
                            <div class="row">
 +
                                <div class="col-md-2 col-sm-2"></div>
 +
 +
                                <div class="col-md-8 col-sm-8">
 +
                                    <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                        <div class="card" style="width: 20rem; display: block; margin: auto">
 +
                                            <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/97/Mingdaojay1003-19.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                        </div>
 +
                                    </div>
 +
                                </div>
 +
 +
                                <div class="col-md-2 col-sm-2"></div>
 +
                            </div>
 +
                            <!-- fifth END -->
 +
                            <!-- Card Group End -->
 +
                            <button class="btn bg-primary text-white" style="letter-spacing: 0.3rem; display: block; margin: auto; margin-bottom: 3rem; margin-top: 3rem">
 +
                                <span style="padding: 0 2rem 0 2rem">Gene cloning for CP29-RBS-aeBlue/pLBA169</span>
 +
                            </button>
 +
                            <!-- Card Group -->
 +
                            <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                <!-- first -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/f/f9/Mingdaojay1003-20.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- second -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/91/Mingdaojay1003-21.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                            </div>
 +
                            <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                <!-- third -->
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/5/5e/Mingdaojay1003-22.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth -->
 +
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/a/a3/Mingdaojay1003-23.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth END -->
 +
                            </div>
 +
                          </div>
 +
                        </div>
 +
                    <!-- New card end -->
 +
 +
                    <!-- New card 2 -->
 +
                        <div class="card" style="width: 100%; margin-top: 3rem; margin-bottom: 3rem">
 +
                            <img src="https://static.igem.org/mediawiki/2017/6/6f/Mingdaojay1003-24.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 45%">
 +
                          <div class="card-body">
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230001</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Type:  </b>Basic part</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                EmR (Erythromycin resistance gene) was amplified from pMG36e vector and cloned onto pSB1C3.
 +
                            </h4>
 +
                            <img src="https://static.igem.org/mediawiki/2017/4/4e/Mingdaojay1003-25.jpeg" style="margin-top:2rem; margin-left: 2.5rem; width: 45%">
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230002</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Type:  </b>Composite part</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                EmR (Erythromycin resistance gene) was amplified from pMG36e vector and cut by XbaI &amp; PstI. Then, the fragment was assembled with RBS/pSB1A3 (BBa_B0034) cut by SpeI &amp; PstI
 +
                            </h4>
 +
                            <button class="btn text-white" style="letter-spacing: 0.3rem;background-color: #004480; display: block; margin: auto; margin-bottom: 3rem; margin-top: 3rem">
 +
                                <span style="padding: 0 1rem 0 1rem">Erythromycin Resistance Gene as a Selection Marker</span>
 +
                            </button>
 +
                            <h4 class="w3-padding-16">
 +
                                Erythromycin is a common selection marker for engineering lactic acid bacteria1. And teams Uppsala and TMMU_China were using it to select the engineered Lactobacillus with erythromycin resistant strains.
 +
                            <h4 class="w3-padding-16">
 +
                                However, the BioBrick parts containing the erythromycin resistance gene are currently unavailable. On the other hand, iGEM HQ is unable to service the request of plasmid carrying erythromycin resistance gene. Maybe iGEM HQ can’t manipulate erythromycin resistant bacteria so far.
 +
                            <h4 class="w3-padding-16">
 +
                                Therefore, we decided to clone the erythromycin resistance gene (EmR) by ourselves. We searched the vector carrying EmR and found an iGEM team HKUST in 2010 working with it. We got the pMG36e vector from the team and cloned the EmR gene out onto pSB1C3. We also made RBS-EmR/pSB1C3 (Bba_K2230002) and RBS-EmR-TT/pSB1C3 (Bba_K2230002) by assembling RBS (B0034) and double terminator (B0015) parts, respectively. These will provide iGEM team this resistance gene as selection marker in the future.
 +
                            </h4>
 +
                            <!-- REFERNCE -->
 +
                            <div class="col-md-12 col-sm-12" >
 +
                                <div style="margin-left: 20px; margin-top: 3rem">
 +
                                    <!-- Box -->
 +
                                    <div style="margin-right: 50px">
 +
                                        <ul class="list-group">
 +
                                          <li class="list-group-item w3-wide" style="text-align: center"><strong>- REFERENCE -</strong></li>
 +
                                          <li class="list-group-item" style="color: black;">1. &nbsp;&nbsp;&nbsp;&nbsp;Genetic engineering techniques for lactic acid bacteria: construction of a stable shuttle vector and expression vector for β-glucuronidase. Biotechnol Lett. 2014;36(2):327-35.</li>
 +
                                        </ul>
 +
                                    </div>
 +
                                </div>
 +
                                <!-- REFERNCE END -->
 +
                            <!-- Card Group -->
 +
                            <div class="card-group" style="margin-right:5rem; margin-left: 5rem; margin-top: 3rem">
 +
 +
                                <div class="row">
 +
                                    <div class="col-md-6 col-sm-6">
 +
                                        <div class="row">
 +
                                            <!-- first -->
 +
                                            <div class="col-md-12 col-sm-12">
 +
                                                <div class="card">
 +
                                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/1/1c/Mingdaojay1003-28.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                                </div>
 +
                                            </div>
 +
                                            <div class="col-md-12 col-sm-12">
 +
                                                <!-- second -->
 +
                                                <div class="card">
 +
                                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/7/7f/Mingdaojay1003-29.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                                </div>
 +
                                            </div>
 +
                                        </div>
 +
                                    </div>
 +
                                    <div class="col-md-6 col-sm-6">
 +
                                        <!-- third -->
 +
                                        <div class="card">
 +
                                        <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/d/d3/Mingdaojay1003-26.jpeg" alt="Card image cap" style="display: block; margin: auto; width: 100%">
 +
                                        </div>
 +
                                    </div>
 +
                                </div>
 +
                                <div class="row">
 +
                                    <div class="col-md-6 col-sm-2"></div>
 +
                                    <div class="col-md-6 col-sm-2"></div>
 +
                                </div>
 +
                            </div>
 +
                            <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                <!-- third -->
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/9c/Mingdaojay1003-27.jpeg" alt="Card image cap" style="display: block; margin: auto; width: 70%">
 +
                                </div>
 +
                                <!-- fourth -->
 +
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/6/60/Mingdaojay1003-30.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth END -->
 +
                            </div>
 +
 +
                            <!-- Part -->
 +
                            <img src="https://static.igem.org/mediawiki/2017/9/9d/Mingdaojay1003-31.jpeg" style="margin-top:2rem; margin-left: 2.5rem; width: 45%" class="w3-padding-24">
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230003</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Type:  </b>Composite part</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                The fragment of RBS-EmR of RBS-EmR/pSB1C3 (BBa_K2230002) cut by EcoRI &amp; SpeI was gel eluted and assembled with TT/pSB1C3 (Double terminator, BBa_B0015) cut by EcoRI &amp; XbaI.
 +
                            </h4>
 +
                            <!-- Part -->
 +
                            <img src="https://static.igem.org/mediawiki/2017/f/f0/Mingdaojay1003-32.jpeg" style="margin-top:2rem; margin-left: 2.5rem; width: 75%" class="w3-padding-24">
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230004</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Type:  </b>Composite part (device)</h3>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4> RBS-EmR which was cut and gel-eluted from RBS-EmR/pSB1C3 (BBa_K2230002) was assembled with CP29-RBS-aeBlue/pLBA169 (BBa_K2230000)
 +
                            </h4>
 +
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function:  </b></h3>
 +
                            <h4> A vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA, which encodes a surface layer protein A. EmR (erythromycin resistance gene), which is driven by the high constitutive promoter of slpA, acts as a selection marker. And aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will be erythromycin resistant and express blue color proteins.
 +
                            </h4>
 +
 +
                            <!-- Card Group -->
 +
                            <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                <!-- first -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/4/4a/Mingdaojay1003-33.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- second -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/a/a9/Mingdaojay1003-35.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                            </div>
 +
                            <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                <!-- third -->
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/0/01/Mingdaojay1003-34.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth -->
 +
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/c/c4/Mingdaojay1003-6.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth END -->
 +
                            </div>
 +
                          </div>
 +
                        </div>
 +
                    <!-- New card 2 end -->
 +
                </div>
 +
            </div>
 +
        </div>
 +
            <!-- Recombination end -->
 +
 +
            <!-- Suicide start -->
 +
            <div id="suicide-md">
 +
                <div class="row w3-padding-32">
 +
                    <div class="col-md-2 col-sm-2" style="background-color: #fff"></div>
 +
                    <div class="col-md-8 col-sm-8" style="background-color: #fff"><img src="https://static.igem.org/mediawiki/2017/9/91/Mingdaojay1003-37.jpeg" width="100%"> </div>
 +
                    <div class="col-md-2 col-sm-2" style="background-color: #fff"></div>
 +
                </div>
 +
            </div>
 +
            <!-- card -->
 +
            <div class="row">
 +
                <div class="col-md-10 col-sm-10" style="display: block; margin: auto; margin-top: 3rem">
 +
                        <div class="card">
 +
                          <h4 class="card-header text-center" style="letter-spacing: 0.3rem; font-size:1.8rem; background-color: #004480; color: white">Background</h4>
 +
                          <div class="card-body">
 +
                            <h4 class="card-title w3-padding-16">  Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo  designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.
 +
                            </h4>
 +
                            <h4 class="card-title w3-padding-16">
 +
                                PI promoter [BBa_K861170] is a modified version to Pcar with correction of -10 position in the promoter region, which gives the promoter stronger activity and a weaker CRP interaction. The PI promoter [BBa_K861170] was used as a major part in the work of team NCKU_Tainan in 2016. These two parts have been used in our work.
 +
                            </h4>
 +
                            <h4 class="card-title w3-padding-16">
 +
                                PhlF repressor system contains the repressor PhlF [BBa_K1725041] and the PhlF repressible promoter [BBa_K1725001] created by Glasgow in 2015. PhlF could repress GFP fluorescence intensity by 83-fold according to the study of Glasgow’s work.
 +
                            </h4>
 +
                            <h4 class="card-title w3-padding-16">
 +
                                We’ve innovated this year a novel glucose responsive repressor system (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 [BBa_K2230012]) by connecting these two system and extend the function of them. Furthermore, based on this new system, we assembled lysis and nuclease genes to the device and created the suicide circuit controlled by the presence of glucose (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP-sRBS-lysis-sRBS-NucA/pSB1C3 [BBa_K2230017]).
 +
                            </h4>
 +
                            <h4 class="card-title w3-padding-16">
 +
                                Lysis gene [BBa_K117000] created by NTU-Singapore in 2008 encodes Lysis protein which could not only lyse bacterial cell membrane but also activate the endonuclease of Colicin E7 (ColE7). The lysis-colicin is one class of bacteriocins which are produced to response to worsening environmental conditions and outcompete other bacteria<sup>1</sup>.
 +
                            </h4>
 +
                            <h4 class="card-title w3-padding-16">
 +
                                NucA [BBa_K1159105] created by TU-Munich in 2013 from Staphylococcus aureus produces a thermostable exo- and endo-nuclease that is able to degrade genomic DNAs2. NucA also has a role in the cleavage of extracellular DNAs and preventing biofilm formation.
 +
                            </h4>
 +
                            <!-- PART start -->
 +
 +
                            <h4 class="card-header text-center" style="letter-spacing: 0.3rem; font-size:1.8rem; background-color: #004480; color: white">The Parts</h4>
 +
                            <div class="row">
 +
                                <div class="col-md-7 col-sm-7">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/0/08/Mingdaojay1003-38.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                    <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230005</h3>
 +
                                    <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                PI-RBS-RFP was amplified from RFP Coding Device (BBa_J04450) using a primer with PI-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                RFP can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 &amp; NCKU_Tainan 2016, the promoter, PI, is glucose responsive.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <!-- PART start -->
 +
                            <div class="row">
 +
                                <div class="col-md-7 col-sm-7">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/38/Mingdaojay1003-39.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                    <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230006</h3>
 +
                                    <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                Pcar-RBS-RFP was amplified from RFP Coding Device (BBa_J04450) using a primer with Pcar-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                RFP can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 &amp; NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <!-- PART start -->
 +
                            <div class="row">
 +
                                <div class="col-md-7 col-sm-7">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/1/1f/Mingdaojay1003-40.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                    <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230007</h3>
 +
                                    <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                PI-RBS-aeBlue was amplified from CP29-RBS-aeBlue (BBa_K1033280) using a primer with PI-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                aeBlue can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 &amp; NCKU_Tainan 2016, the promoter, PI, is glucose responsive.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <!-- PART start -->
 +
                            <div class="row">
 +
                                <div class="col-md-7 col-sm-7">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/4/4d/Mingdaojay1003-41.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                    <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230008</h3>
 +
                                    <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                Pcar-RBS-aeBlue was amplified from CP29-RBS-aeBlue (BBa_K1033280) using a primer with Pcar-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                aeBlue can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 &amp; NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <!-- Card Group -->
 +
                            <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                <!-- first -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/8/88/Mingdaojay1003-42.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- second -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/e/e7/Mingdaojay1003-43.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                            </div>
 +
                            <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                <!-- third -->
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/6/6a/Mingdaojay1003-44.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth -->
 +
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/4/46/Mingdaojay1003-45.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth END -->
 +
                            </div>
 +
                            <!-- Card Group end -->
 +
                            <hr style="width: 100%; color: #0c4a7c; background-color: #0c4a7c">
 +
 +
                            <!-- PART start -->
 +
                            <div class="row">
 +
                                <div class="col-md-7 col-sm-7">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/3d/Mingdaochuck1003-46.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                    <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230009</h3>
 +
                                    <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                PI-RBS-PhlF-T was amplified from RBS + PhlF repressor + terminator (BBa_K1725041) using a primer with PI-RBS (B0032) sequence. The PCR product was ligated to pSB1C3 cut by XbaI and PstI.
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                Repressor PhlF can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 &amp; NCKU_Tainan 2016, the promoter, PI, is glucose responsive.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <!-- PART start -->
 +
                            <div class="row">
 +
                                <div class="col-md-7 col-sm-7">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/fc/Mingdaochuck1003-47.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                    <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K22300010</h3>
 +
                                    <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                            <h4>
 +
                                  Pcar-RBS-PhlF was amplified from RBS + PhlF repressor + terminator (BBa_K1725041) using a primer with Pcar-RBS (B0032) sequence. The PCR product was ligated to pSB1C3 cut by XbaI and PstI.
 +
                            </h4>
 +
                            <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                            <h4>
 +
                                Repressor PhlF can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 &amp; NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.
 +
                            </h4>
 +
                            <!-- PART end -->
 +
                            <!-- Card Group -->
 +
                            <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                <!-- first -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/b/b3/Mingdaochuck1003-48.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- second -->
 +
                                <div class="card">
 +
                                <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/6/60/Mingdaochuck1003-49.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                            </div>
 +
                            <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                <!-- third -->
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/98/Mingdaochuck1003-50.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth -->
 +
 +
                                <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/3/38/Mingdaochuck1003-51.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                </div>
 +
                                <!-- fourth END -->
 +
                              </div>
 +
                              <!-- Card Group end -->
 +
 +
                              <hr style="width: 100%; color: #0c4a7c; background-color: #0c4a7c">
 +
 +
                              <!-- PART start -->
 +
                              <div class="row">
 +
                                  <div class="col-md-7 col-sm-7">
 +
                                      <img src="https://static.igem.org/mediawiki/2017/8/83/Mingdaochuck1003-52.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                  </div>
 +
                                  <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                      <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230011</h3>
 +
                                      <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                  </div>
 +
                              </div>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                              <h4>
 +
                                  Pr-sRBS-GFP (BBa_K1725001) with strong RBS (B0034) was assembled with PI-RBS-PhlF-T/pSB1C3 (BBa_K2230009).
 +
                              </h4>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                              <h4>
 +
                                  The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.
 +
                              </h4>
 +
                              <!-- PART end -->
 +
                              <!-- PART start -->
 +
                              <div class="row">
 +
                                  <div class="col-md-7 col-sm-7">
 +
                                      <img src="https://static.igem.org/mediawiki/2017/6/6b/Mingdaochuck1003-53.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                  </div>
 +
                                  <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                      <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230012</h3>
 +
                                      <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                  </div>
 +
                              </div>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                              <h4>
 +
                                  Pr-sRBS-GFP (BBa_K1725001) with strong RBS (B0034) was assembled with Pcar-RBS-PhlF-T/pSB1C3 (BBa_K2230010)
 +
                              </h4>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                              <h4>
 +
                                  The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.
 +
                              </h4>
 +
                              <!-- PART end -->
 +
 +
                              <!-- PART start -->
 +
                              <div class="row">
 +
                                  <div class="col-md-7 col-sm-7">
 +
                                      <img src="https://static.igem.org/mediawiki/2017/d/d1/Mingdaochuck1003-54.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                  </div>
 +
                                  <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                      <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230013</h3>
 +
                                      <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                  </div>
 +
                              </div>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                              <h4>
 +
                                  Pr-wRBS-GFP (BBa_K1725002) with weak RBS (B0032) was assembled with PI-RBS-PhlF-T/pSB1C3 (BBa_K2230009)
 +
                              </h4>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                              <h4>
 +
                                  The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.
 +
                              </h4>
 +
                              <!-- PART end -->
 +
 +
                              <!-- PART start -->
 +
                              <div class="row">
 +
                                  <div class="col-md-7 col-sm-7">
 +
                                      <img src="https://static.igem.org/mediawiki/2017/b/b7/Mingdaochuck1003-55.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 75%">
 +
                                  </div>
 +
                                  <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                      <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230014</h3>
 +
                                      <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                  </div>
 +
                              </div>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                              <h4>
 +
                                  Pr-wRBS-GFP (BBa_K1725002) with strong RBS (B0034) was assembled with Pcar-RBS-PhlF-T/pSB1C3 (BBa_K2230010)
 +
                              </h4>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                              <h4>
 +
                                  The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.
 +
                              </h4>
 +
                              <!-- PART end -->
 +
 +
                              <!-- Card Group -->
 +
                              <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                  <!-- first -->
 +
                                  <div class="card">
 +
                                  <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/7/78/Mingdaochuck1003-56.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                                  <!-- second -->
 +
                                  <div class="card">
 +
                                  <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/9/9a/Mingdaochuck1003-57.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                              </div>
 +
                              <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                  <!-- third -->
 +
                                  <div class="card">
 +
                                      <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/e/e8/Mingdaochuck1003-58.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                                  <!-- fourth -->
 +
 +
                                  <div class="card">
 +
                                      <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/0/09/Mingdaochuck1003-59.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                                  <!-- fourth END -->
 +
                              </div>
 +
                              <!-- Card Group end -->
 +
 +
 +
                              <hr style="width: 100%; color: #0c4a7c; background-color: #0c4a7c">
 +
 +
                              <!-- PART start -->
 +
                              <div class="row">
 +
                                  <div class="col-md-7 col-sm-7">
 +
                                      <img src="https://static.igem.org/mediawiki/2017/f/fd/Mingdaochuck1003-60.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 90%">
 +
                                  </div>
 +
                                  <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                      <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230015</h3>
 +
                                      <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                  </div>
 +
                              </div>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                              <h4>
 +
                                  wRBS-lysis was amplified from Lysis gene (BBa_K117000) using a primer with weak RBS sequence (B0032) and assembled with Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 (BBa_K2230012)
 +
                              </h4>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                              <h4>
 +
                                  Suicide genes of lysis was driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.
 +
                              </h4>
 +
                              <!-- PART end -->
 +
                              <!-- PART start -->
 +
                              <div class="row">
 +
                                  <div class="col-md-7 col-sm-7">
 +
                                      <img src="https://static.igem.org/mediawiki/2017/2/2a/Mingdaochuck1003-61.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 90%">
 +
                                  </div>
 +
                                  <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                      <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230016</h3>
 +
                                      <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                  </div>
 +
                              </div>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                              <h4>
 +
                                  sRBS-lysis was amplified from Lysis gene (BBa_K117000) using a primer with strong RBS sequence (B0034) and assembled with Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 (BBa_K2230012)
 +
                              </h4>
 +
                              <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                              <h4>
 +
                                  Suicide genes of lysis was driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.
 +
                              </h4>
 +
                              <!-- PART end -->
 +
 +
                              <!-- Card Group -->
 +
                              <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                  <!-- first -->
 +
                                  <div class="card">
 +
                                  <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/2/23/Mingdaochuck1003-62.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                                  <!-- second -->
 +
                                  <div class="card">
 +
                                  <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/8/88/Mingdaochuck1003-65.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                              </div>
 +
                              <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                  <!-- third -->
 +
                                  <div class="card">
 +
                                      <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/d/d7/Mingdaochuck1003-63.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                                  <!-- fourth -->
 +
 +
                                  <div class="card">
 +
                                      <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/d/dd/Mingdaochuck1003-64.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                  </div>
 +
                                  <!-- fourth END -->
 +
                                </div>
 +
                                <!-- Card Group end -->
 +
 +
                                <hr style="width: 100%; color: #0c4a7c; background-color: #0c4a7c">
 +
 +
 +
                                <!-- PART start -->
 +
                                <div class="row">
 +
                                    <div class="col-md-7 col-sm-7">
 +
                                        <img src="http:/2017.igem.org/wiki/images/d/d5/Mingdaochuck1003-66.jpeg" style="margin-top:2rem; margin-left: 3rem; width: 90%">
 +
                                    </div>
 +
                                    <div class="col-md-5 col-sm-5" style="padding-top: 3rem">
 +
                                        <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Part No.  :</b> BBa_K2230017</h3>
 +
                                        <h3 class="card-title" style="letter-spacing: 0.1rem"><b>Type:  </b>Composite part (device)</h3>
 +
                                    </div>
 +
                                </div>
 +
                                <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Cloning:  </b></h3>
 +
                                <h4>
 +
                                    sRBS-NucA was amplified from Mature Nuclease NucA from Staphylococcus aureus (BBa_K1159105) using a primer with strong RBS sequence (B0034) and assembled with Pcar-RSB-PhlF-T-Pr-sRBS-GFP-sRBS-lysis/pSB1C3
 +
                                </h4>
 +
                                <h3 class="card-title" style="letter-spacing: 0.2rem"><b>Function: </b></h3>
 +
                                <h4>
 +
                                    Suicide genes of lysis and nuclease (NucA) were driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.
 +
                                </h4>
 +
                                <!-- PART end -->
 +
 +
                                <!-- Card Group -->
 +
                                <div class="card-group" style="margin-right:5rem; margin-left: 5rem">
 +
                                    <!-- first -->
 +
                                    <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/6/64/Mingdaochuck1003-67.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                    </div>
 +
                                    <!-- second -->
 +
                                    <div class="card">
 +
                                    <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/a/a5/Mingdaochuck1003-68.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                    </div>
 +
                                </div>
 +
                                <div class="card-group" style="margin-right: 5rem; margin-left: 5rem">
 +
                                    <!-- third -->
 +
                                    <div class="card">
 +
                                        <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/a/a1/Mingdaochuck1003-69.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                    </div>
 +
                                    <!-- fourth -->
 +
 +
                                    <div class="card">
 +
                                        <img class="card-img-top" src="https://static.igem.org/mediawiki/2017/1/1a/Mingdaochuck1003-70.jpeg" alt="Card image cap" style="display: block; margin: auto">
 +
                                    </div>
 +
                                    <!-- fourth END -->
 +
                                </div>
 +
                                <!-- Card Group end -->
 +
 +
                                <h4 class="card-header text-center" style="letter-spacing: 0.3rem; font-size:1.8rem; background-color: #004480; color: white; margin-top: 5rem">Experiment and Results</h4>
 +
 +
                                <button class="btn bg-primary text-white" style="letter-spacing: 0.3rem; margin-top: 2rem; margin-bottom: 3rem; margin-left: 3rem;">
 +
                                  <span class="badge badge-info">1</span>
 +
                                  &nbsp;&nbsp;&nbsp;Glucose responsive promoter activity
 +
                                  <span class="sr-only"></span>
 +
                                </button>
 +
 +
                                <img src="https://static.igem.org/mediawiki/2017/8/82/MD-P28-TOP.png" width="60%" style="padding-left: 2.5rem">
 +
                                <br><br>
 +
                                <h4 style="padding-left: 3rem">The result shown in Fig. 2 indicated that PI promoter has significant activity in LB culture media. However, the activity of Pcar promoter is greater than negative control but much smaller than PI and positive control. It’s consistent with the properties of PI and Pcar promoters just mentioned previously in GENE CLONING section and described previously in Part Resgistry [Part: BBa_K861170] by team WHU-China in 2012 who designed the promoters.</h4>
 +
 +
                                <h4 style="padding-left: 3rem"> In our experiment as presented in Fig. 3, PI and Pcar promoters just responded to various concentrations of glucose with a  very slight dose-dependent increase. This phenomenon didn’t correspond to the data provided by team WHU-China in 2012 and team NCKU_Tainan in 2016. Maybe our measurement was not in an optimized condition. Or the reporter of RFP activity was not sensitive enough to respond this difference.</h4>
 +
                                <div class="row">
 +
 +
                                    <div class="col-md-6 col-sm-6">
 +
        <img src="https://static.igem.org/mediawiki/2017/9/99/MD-P20-bl.png" style="padding-left:3rem;float:right" width="75%">
 +
        </div>
 +
        <div class="col-md-6 col-sm-6">
 +
        <img src="https://static.igem.org/mediawiki/2017/9/9e/MD-P20-br.png" width="70%">
 +
        </div>
 +
 +
        <div class="col-md-12 col-sm-12" style="display:block; margin: auto">
 +
 +
        <button class="btn bg-primary text-white" style="letter-spacing: 0.3rem; margin-left: 3rem; margin-top: 5rem; margin-bottom: 3rem; background-color:#004480">
 +
            <span class="badge badge-info">2</span>
 +
                &nbsp;Functional assay for Glucose Responsive Suppressor System
 +
            <span class="sr-only"></span>
 +
        </button>
 +
 +
        <img src="https://static.igem.org/mediawiki/2017/1/17/Mingdaochuck1017-11.png" width="60%" style="padding-left: 4.5rem; margin-bottom: 3rem">
 +
 +
        <h4 style="padding-left: 5rem">
 +
                                                  The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD<sub>600</sub> was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. 100 μl of the bacterial culture was put into one well of a black-walled, clear-bottom 96-well microplates (Thermo Fisher Scientific Inc.). The fluorescence intensity was measured using BioTek Synergy H1 Hybrid Multi-Mode Reader System at Ex/Em = 488nm/518nm  for GFP.
 +
                                        </h4>
 +
 +
                                    <img src="https://static.igem.org/mediawiki/2017/9/9c/Mingdaophil1014-1.jpeg" width="60%" style="padding-left: 4.5rem; margin-bottom: 3rem">
 +
 +
        <h4 style="padding-left: 5rem">In the assay for repressor system, the data in Fig. 2 gave the similar results as team Glasgow did in 2015, in which the strong activity of the repressible promoter was significantly repressed in the presence of PhlF repressor.
 +
                                        </h4>
 +
                                        <h4 style="padding-left: 5rem">
 +
                                            In the assay for glucose responsive repressor system, we improved the Glasgow’s BioBrick device, in which the expression of PhlF repressor was driven by a glucose response promoter, Pcar. The result in Fig 3 clearly indicated that the GFP activity driven by the repressible promoter was gradually increased in response to the loss of glucose to 1.88 folds compared to the initial GFP activity at the beginning culture in M9 media, suggesting that the level of expression of PhlF was positively corresponding to the concentration of glucose.
 +
                                        </h4>
 +
        </div>
 +
                                    <div class="col-md-6 col-sm-6">
 +
                                        <img src="https://static.igem.org/mediawiki/2017/4/4d/Mingdaophil1014-2.jpeg" width="100%" style="padding-left: 4.5rem; margin-bottom: 3rem">
 +
                                    </div>
 +
                                    <div class="col-md-6 col-sm-6">
 +
                                        <img src="https://static.igem.org/mediawiki/2017/3/35/Mingdaophil1014-3.jpeg" width="100%" style="padding-left: 4.5rem; margin-bottom: 3rem">
 +
                                    </div>
 +
                                    <div class="col-md-10 col-sm-10" style="display: block; margin: auto">
 +
                                        <img src="https://static.igem.org/mediawiki/2017/f/f1/Mingdaophil1014-4.jpeg" width="100%" style="padding-left: 4.5rem; margin-bottom: 3rem">
 +
                                    </div>
 +
                                </div>
 +
                                <div class="row">
 +
                                    <div class="col-md-12 col-sm-12">
 +
                                        <button class="btn bg-primary text-white" style="letter-spacing: 0.3rem; margin-top: 2rem; margin-bottom: 3rem; margin-left: 3rem;">
 +
                                          <span class="badge badge-info">3</span>
 +
                                          &nbsp;&nbsp;&nbsp;Functional assay for glucose responsive suicide circuit
 +
                                          <span class="sr-only"></span>
 +
                                        </button>
 +
 +
                                        <img src="https://static.igem.org/mediawiki/2017/d/d5/Mingdaochuck1003-66.jpeg" width="60%" style="padding-left: 2.5rem">
 +
                                        <br><br>
 +
                                        <h4 style="padding-left: 3rem">
 +
                                                The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD<sub>600</sub> was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. 100 μl of the bacterial culture was put into one well of a black-walled, clear-bottom 96-well microplates (Thermo Fisher Scientific Inc.). OD<sub>600</sub> was measured using BioTek Synergy H1 Hybrid Multi-Mode Reader System. Further,  the culture media were taken out and diluted 106 times following by spreading onto LB Cm agar plate at 37°C overnight. The third day, the numbers of colonies were counted and bacterial viability was calculated.
 +
                                        </h4>
 +
                                        <img src="https://static.igem.org/mediawiki/2017/f/fa/Mingdaophil1014-5.jpeg" width="70%" style="display: block; margin: auto">
 +
 +
                                        <h4 style="padding-left: 3rem">
 +
                                        As you can see in Fig 7., the OD value in response to the decreasing concentration of glucose was gradually reduced to 1.89 much less than average 2.71 in control group without suicide gene expression, implying that the suicide proteins killed the cells in the loss of glucose in the environment. Moreover, when the bacteria were grown in M9 media with 0.5mM glucose for 4 hours, the survival rate was decreased to 34% compared to 56% of bacteria without suicide genes (Fig. 8). And the cell numbers were reduced to 671 compared to 1120 of bacteria without suicide genes. Both data confirmed that this suicide device works well and indicated that killing process began when glucose in the media was running out.
 +
                                        </h4>
 +
                                    </div>
 +
                                    <div class="col-md-6 col-sm-6">
 +
                                        <img src="https://static.igem.org/mediawiki/2017/0/0a/Mingdaophil1014-6.jpeg" width="70%" style="display: block; margin: auto">
 +
                                    </div>
 +
                                    <div class="col-md-6 col-sm-6">
 +
                                        <img src="https://static.igem.org/mediawiki/2017/4/43/Mingdaophil1014-7.jpeg" width="80%" style="display: block; margin: auto">
 +
                                    </div>
 +
                                    <div class="col-md-12 col-sm-12">
 +
                                        <img src="https://static.igem.org/mediawiki/2017/8/8f/Mingdaophil1014-8.jpeg" width="80%" style="display: block; margin: auto">
 +
                                    </div>
 +
                                </div>
 +
                        </div>
 +
                </div>
 +
            </div>
 +
            <!-- CSMU END -->
 +
            <!-- Suicide end -->
 +
            <!-- Transporter start -->
 +
            <div id="transport-md">
 +
                <div class="row w3-padding-32">
 +
                    <div class="col-md-2 col-sm-2" style="background-color: #fff"></div>
 +
                    <div class="col-md-8 col-sm-8" style="background-color: #fff"><img src="https://static.igem.org/mediawiki/2017/9/9e/Mingdaochuck1003-71.jpeg" width="100%"> </div>
 +
                    <div class="col-md-2 col-sm-2" style="background-color: #fff"></div>
 +
                </div>
 +
            </div>
 +
            <!-- Transporter end -->
 +
 +
            <!-- CSMU -->
 +
            <div class="row">
 +
                <div class="col-md-10 col-sm-10" style="display: block; margin: auto; margin-top: 3rem">
 +
                        <div class="card">
 +
                          <h4 class="card-header text-center" style="letter-spacing: 0.3rem; font-size:1.8rem; background-color: #004480; color: white">Background</h4>
 +
                          <div class="card-body">
 +
                            <h4 class="card-title w3-padding-32"> Salmonella typhimurium LT2 has two glucose-specific transporter systems, PTS system and sodium/glucose cotransporter. PTS system contains two subunits IIA encoded by crr and IIBC by ptsG which are assembled to a high-affinity active transporter. The other is a Na+/glucose cotransporter encoded by STM1128 that contributes to facilitated transport with lower glucose affinity. Based on our research, the glucose transporter of Salmonella has a lower Km compared to human small intestine, Staphylococcus and E. coli (Table 1), indicating a higher efficiency for glucose uptake. In our project, we created the glucose transporter device and genetically engineer microbes with these two systems.</h4>
 +
                            <img src="https://static.igem.org/mediawiki/2017/5/5d/MD-Just-a-table.png" width="60%" style="display: block; margin: auto">
 +
                            <h4 class="card-title w3-padding-32">
 +
                                In order to express the genes in <i>E. coli</i> for demonstration and in probiotics for proof-of-concept in a real world. We chose promoter CP29 that is a strong constitutive promoter working well in both <i>E. coli</i> and Lactobacillus spp<sup>1</sup>. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280) was used and to be assembled with the transporter genes.
 +
                            </h4>
 +
                            <!-- Part 1-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/3d/Mingdaochuck1003-72.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230018</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Basic Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The crr gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3 </h4>
 +
                            <!-- Part end -->
 +
 +
                            <!-- Part 2-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/3/3d/Mingdaochuck1003-72.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230019</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The crr gene was amplified from gDNA of Salmonella typhimurium using a primer with RBS sequence (B0034), and then cloned onto pSB1C3  </h4>
 +
                            <!-- Part end -->
 +
 +
                            <!-- Part 3-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/9/9a/Mingdaochuck1003-74.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230020</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Basic Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The ptsG gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3  </h4>
 +
                            <!-- Part end -->
 +
 +
                            <!-- Part 4-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/5/50/Mingdaochuck1003-75.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230021</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The ptsG gene was amplified from gDNA of Salmonella typhimurium using a primer with RBS sequence (B0034), and then cloned onto pSB1C3 </h4>
 +
                            <!-- Part end -->
 +
 +
                            <!-- Part 5-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/a/ad/Mingdaochuck1003-76.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230022</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Basic Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3 </h4>
 +
                            <!-- Part end -->
 +
                            <!-- Part 6-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/6/63/Mingdaochuck1003-77.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230023</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The STM1128 gene was amplified from gDNA of Salmonella typhimurium using a primer with RBS sequence (B0034), and then cloned onto pSB1C3 </h4>
 +
                            <!-- Part end -->
 +
                            <div class="row" style="margin-top: 6rem">
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/5/53/Mingdaochuck1003-78.jpeg" width="100%">
 +
                                </div>
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/9/9a/Mingdaochuck1003-79.jpeg" width="100%">
 +
                                </div>
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/f/fd/Mingdaochuck1003-80.jpeg" width="100%">
 +
                                </div>
 +
                            </div>
 +
                            <div class="row">
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/9/98/Mingdaochuck1003-81.jpeg" width="100%">
 +
                                </div>
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/4/49/Mingdaochuck1003-82.jpeg" width="100%">
 +
                                </div>
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/6/68/Mingdaochuck1003-83.jpeg" width="100%">
 +
                                </div>
 +
                            </div>
 +
 +
                            <!-- Part 7-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/b/bb/Mingdaochuck1003-84.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230024</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The RBS-ptsG part (BBa_K2230022) was assembled with Double Terminator/pSB1C3 (BBa_B0015)</h4>
 +
                            <!-- Part end -->
 +
                            <!-- Part 8-->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/a/a3/Mingdaochuck1003-85.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 3rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230025</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The RBS-STM1128 part (BBa_K2230024) was assembled with Double Terminator/pSB1C3 (BBa_B0015)</h4>
 +
                            <!-- Part end -->
 +
                            <div class="row" style="margin-top: 3rem">
 +
                                <div class="col-md-3 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/1/10/Mingdaochuck1003-86.jpeg" width="100%">
 +
                                </div>
 +
                                <div class="col-md-5 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/5/5a/Mingdaochuck1003-87.jpeg" width="100%">
 +
                                </div>
 +
                                <div class="col-md-4 col-sm-4">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/8/8d/Mingdaochuck1003-88.jpeg" width="100%">
 +
                                </div>
 +
                            </div>
 +
                            <!-- Part -->
 +
                            <div class="row" style="margin-top: 3rem">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/4/45/Mingdaochuck1003-89.jpeg" width="90%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 1rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230026</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">The RBS-crr part (BBa_K2230019) was assembled with RBS-ptsG-TT/pSB1C3 (BBa_K2230024)</h4>
 +
                            <!-- Part end -->
 +
                            <!-- Part -->
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/b/be/Mingdaochuck1003-90.jpeg" width="100%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 1rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230027</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">Promoter CP29-RBS-aeBlue (BBa_K1033280) was assembled with RBS-crr-RBS-ptsG-TT/pSB1C3 (BBa_K2230026) </h4>
 +
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Function:</b></h3>
 +
                            <h4 style="padding-left: 5rem">An active high-affinity glucose transporter system. Promoter CP29 is a constitutive and strong promoter which works well in both E. coli and Lactobacillus spp. The blue protein encoded by aeBlue gene acts as indicator for the gene expression in the transgenic strain. This device can help E. coli and Lactobacillus spp. efficiently take up and retrieve glucose in the environment. </h4>
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <h4>
 +
                                        <img src="https://static.igem.org/mediawiki/2017/5/55/Mingdaochuck1003-91.jpeg" width="70%" style="display: block; margin: auto">
 +
                                    </h4>
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <h4>
 +
                                        <img src="https://static.igem.org/mediawiki/2017/1/14/Mingdaochuck1003-92.jpeg" width="70%" style="display: block; margin: auto">
 +
                                    </h4>
 +
                                </div>
 +
                            </div>
 +
                            <!-- Part end -->
 +
 +
                            <!-- Part -->
 +
                            <div class="row" style="margin-top: 3rem">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <img src="https://static.igem.org/mediawiki/2017/e/e6/Mingdaochuck1003-93.jpeg" width="100%" style="padding-left: 2.5rem">
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6" style="padding-top: 1rem">
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Part No.:</b> BBa_K2230028</h3>
 +
                                    <h3 style="padding-left: 3rem; letter-spacing: 0.2rem"><b>Type:</b> Composite Part (device)</h3>
 +
                                </div>
 +
                            </div>
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Cloning:</b></h3>
 +
                            <h4 style="padding-left: 5rem">Promoter CP29-RBS-aeBlue (BBa_K1033280) was assembled with RBS-STM1128-TT/pSB1C3 (BBa_K2230025) </h4>
 +
 +
                            <h3 style="padding-left: 5rem; letter-spacing: 0.2rem"><b>Function:</b></h3>
 +
                            <h4 style="padding-left: 5rem">
 +
                                A facilitated low-affinity glucose transporter system. Promoter CP29 is a constitutive and strong promoter which works well in both E. coli and Lactobacillus spp. The blue protein encoded by aeBlue gene acts as indicator for the gene expression in the transgenic strain. This device can help E. coli and Lactobacillus spp. efficiently take up and retrieve glucose in the environment.
 +
                            </h4>
 +
                            <div class="row">
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <h4>
 +
                                        <img src="https://static.igem.org/mediawiki/2017/8/8c/Mingdaochuck1003-94.jpeg" width="90%" style="display: block; margin: auto">
 +
                                    </h4>
 +
                                </div>
 +
                                <div class="col-md-6 col-sm-6">
 +
                                    <h4>
 +
                                        <img src="https://static.igem.org/mediawiki/2017/4/42/Mingdaochuck1003-95.jpeg" width="80%" style="display: block; margin: auto">
 +
                                    </h4>
 +
                                </div>
 +
                            </div>
 +
                            <!-- Part end -->
 +
 +
                            <h4 class="card-header text-center" style="letter-spacing: 0.3rem; font-size:1.8rem; background-color: #004480; color: white; margin-top: 5rem">Experiment and Results</h4>
 +
                            <!-- TEST -->
 +
                                <div id="test1019" style="padding: 3rem 3rem 3rem 3rem ">
 +
                                    <!-- Box -->
 +
                                                <div class="box" style="background-color: rgba(255, 255, 255, 0); margin-right: 50px">
 +
                                                    <h4 style="font-color: #a6a6a6;">To measure glucose uptake by the engineered E. coli expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34μg/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD<sub>600</sub> = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.</h4>
 +
                        <div class="row">
 +
                        <div class="col-md-6 col-sm-12">
 +
                        <br><br>
 +
                        <img src=" https://static.igem.org/mediawiki/2017/7/76/Mingdaophil1019-6.png" width="100%" >
 +
                        <div class="row">
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <h4 style="text-align: center; padding-top: 2rem;"></h4>
 +
                        </div>
 +
                        </div>
 +
                        </div>
 +
                        <div class="col-md-6 col-sm-12">
 +
                        <img src="https://static.igem.org/mediawiki/2017/6/69/Mingdaophil1019-9.png" width="70%" style="display: block; margin: auto;">
 +
                        </div>
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <h4>Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.</h4>
 +
                        </div>
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <img src="https://static.igem.org/mediawiki/2017/a/a7/Mingdaophil1019-8.png" width="55%" style="display: block; margin: auto;">
 +
                        </div>
 +
 +
                        <!-- Result Section -->
 +
                        <div class="col-md-12 col-sm-12" style="margin-top: 5rem">
 +
                        <span class="w3-center w3-padding-small w3-black w3-xlarge w3-wide w3-animate-opacity"> <span class="w3-hide-small"></span> </span>
 +
                                                <span class="w3-center w3-padding-large w3-wide w3-xlarge w3-animate-opacity">Result</span>
 +
                                                <br/><br/>
 +
                        </div>
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <h4>As shown in Fig. 3, the growth of E. coli expressing the PTS reporter (i.e., crr and ptsG genes) was seriously retardant. As I mentioned earlier, the E. coli overexpressing glucose transporter may lose viability because of the toxic metabolites produced in glycolytic pathway. Not surprisingly, the PTS overexpression bacteria was almost unable to absorb glucose. </h4>
 +
                        </div>
 +
                        <!-- Fig 3. -->
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <img src="https://static.igem.org/mediawiki/2017/a/a1/Mingdaophil1019-1.png" width="70%" style="display: block; margin: auto;">
 +
                        </div>
 +
                        <!-- caption of fig -->
 +
                        <div class="row">
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <span style="padding-left: 6rem"><strong>Fig 3. The cell growth overnight and glucose uptake of E. coli expressing the PTS transporter in 4 hours </strong></span>
 +
                        </div>
 +
                        </div>
 +
                        <!-- caption of fig END -->
 +
                        <div class="col-md-12 col-sm-12" style="padding-top: 3rem; padding-bottom: 3rem">
 +
                        <h4>Fig. 4 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3rd hour. The glucose uptake efficiency was achieved upto 97% in Na+/Glu group and greater than in control group with 1.2 times difference.</h4>
 +
                        </div>
 +
                        <!-- Fig 4. ROW -->
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <img src="https://static.igem.org/mediawiki/2017/5/5e/Mingdaophil1019-3.png" width="70%" style="display: block; margin: auto;">
 +
                        </div>
 +
 +
                        <!-- Fig 4. ROW END -->
 +
                        <!-- caption of fig -->
 +
                        <div class="row">
 +
                        <div class="col-md-12 col-sm-12">
 +
                        <strong style="padding-left: 6rem">Fig 4. The cell growth overnight and glucose uptake of E. coli expressing the Na+/Glu transporter at different time point</strong>
 +
                        </div>
 +
                        </div>
 +
                        <!-- caption of fig END -->
 +
                                                        <!-- Result Section -->
 +
                                                        <!-- Discussion Section -->
 +
                                                        <div class="row">
 +
                                                            <div class="col-md-12 col-sm-12 w3-padding" style="display: block; margin: auto">
 +
                                                                    <div class="card mb-3 bg-primary">
 +
                                                                          <div class="card-header text-center"><strong style="color: #fff; letter-spacing:0.5rem;">Discussion</strong></div>
 +
                                                                          <div class="card-body bg-light">
 +
                                                                            <br>
 +
                                                                            <h4>We’ve successfully genetically engineered E. coli expressing high-affinity active PTS transporter system and low-affinity facilitated Na+/glucose cotransporter system by screening in MacConkey and glycerol agar plate with chloramphenicol, respectively. Our data is consistent with the previous study that overexpressing glucose transporter genes do really harm E. coli partly due to disturbing glycolytic pathways.</h4>
 +
                                                                            <h4>PTS expressing bacteria grew difficultly in LB broth but Na+/glucose transporter expressing bacteria grew comparably with general E. coli, suggesting that high-affinity active transporter did interact with sugar-related substances in LB culture media and harm the bacterial survival.</h4>
 +
                                                                            <h4>In our results, glucose absorption by Na+/glucose transporter expressing bacteria was achieved to 97% after 4 hours with 1.2 times enhanced efficiency compared to the normal E. coli. To further increase the rate of glucose uptake, one may think about the glucose metabolism or conversion to other materials when entering into the cell.</h4>
 +
                                                                          </div>
 +
                                                                    </div>
 +
                                                            </div>
 +
                                                        </div>
 +
                                                        <!-- Discussion Section END -->
 +
                        </div>
 +
                                                </div>
 +
                                </div>
 +
                            <!-- TEST -->
 +
                            <div class="row">
 +
                                <!-- REFERNCE -->
 +
                                <div class="col-md-12 col-sm-12" >
 +
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                                              <li class="list-group-item w3-wide" style="text-align: center"><strong>- REFERENCE -</strong></li>
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                                              <li class="list-group-item" style="color: black;">1. &nbsp;&nbsp;&nbsp;&nbsp;Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli. J Gen Microbiol. 1992;138(10):2007-14. </li>
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{{Team:Mingdao}}

Revision as of 06:06, 19 October 2017

iGEM Human Practice

Home Achievements Project Human-Practice Notebook Attributions

Overview

Background

To engineer Lactobacillus acidophilus by chromosome integration through homologous recombination, the selection of integration site is very important for successful recombination and not disturbing bacterial internal metabolism.

Based on the method created by Grace L. Douglas and Todd R. Klaenhammer1, the region between slpA gene (LBA0169) stop codon and the terminator was chosen as the intergenic insertion location. The gene of slpA encodes a surface-layer protein with a strong constitutive promoter activity, which can also drive the expression of the inserted gene.

Fig 1. The schematic diagram of gene integration location (modified from Appl Environ Microbiol. 2011)

To make pLBA169, the downstream region of LBA0169 and the upstream region of the terminator were amplified by PCR. Then, the two PCR-amplified DNA fragments were ligated to pSB1C3 by tripartite ligation.

Next, CP29-RBS-aeBlue was cut from Part: BBa_K1033280. And this gel isolated DNA fragment was further inserted between two recombination regions on pSB1C3. This process created the standard EcoRI-XbaI-PART-SpeI-PstI assembly position.

To delete the extra SpeI recognition site within the one of the recombination region, we performed site-directed mutagenesis to change one nucleotide of SpeI recognition sequence. The final product has been confirmed by DNA sequencing.

You can refer to the following flow chart of gene cloning of the recombination vector.

  • - REFERENCE -
  • 1.     Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM. Appl Environ Microbiol. 2011;77(20):7365-71.
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Part No. : BBa_K2230000

Type: Composite part (device)

Cloning:

Firstly, the elements of upstream and downstream recombination sites were replicated from gDNA of Lactobacillus acidophilus and put onto pSB1C3 as a L. acidophilus recombination vector, named pLBA169, at EcoRI and PstI locations. Secondly, to generate as a standard BioBrick part and assembly vector, the part of Promoter CP29-RBS-aeBlue (BBa_K1033280) was cut by EcoRI and PstI and assembled with the vector. Finally, because there’s a SpeI recognition sequence within the 169dn element, site-directed mutagenesis was performed to change a nucleotide to leave a single SpeI site in the BioBrick suffix region. (See the flow chart and data below).

Function:

A vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA (LBA0169), which encodes a surface layer protein A. The aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will express blue color proteins.

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Part No. : BBa_K2230001

Type: Basic part

Cloning:

EmR (Erythromycin resistance gene) was amplified from pMG36e vector and cloned onto pSB1C3.

Part No. : BBa_K2230002

Type: Composite part

Cloning:

EmR (Erythromycin resistance gene) was amplified from pMG36e vector and cut by XbaI & PstI. Then, the fragment was assembled with RBS/pSB1A3 (BBa_B0034) cut by SpeI & PstI

Erythromycin is a common selection marker for engineering lactic acid bacteria1. And teams Uppsala and TMMU_China were using it to select the engineered Lactobacillus with erythromycin resistant strains.

However, the BioBrick parts containing the erythromycin resistance gene are currently unavailable. On the other hand, iGEM HQ is unable to service the request of plasmid carrying erythromycin resistance gene. Maybe iGEM HQ can’t manipulate erythromycin resistant bacteria so far.

Therefore, we decided to clone the erythromycin resistance gene (EmR) by ourselves. We searched the vector carrying EmR and found an iGEM team HKUST in 2010 working with it. We got the pMG36e vector from the team and cloned the EmR gene out onto pSB1C3. We also made RBS-EmR/pSB1C3 (Bba_K2230002) and RBS-EmR-TT/pSB1C3 (Bba_K2230002) by assembling RBS (B0034) and double terminator (B0015) parts, respectively. These will provide iGEM team this resistance gene as selection marker in the future.

  • - REFERENCE -
  • 1.     Genetic engineering techniques for lactic acid bacteria: construction of a stable shuttle vector and expression vector for β-glucuronidase. Biotechnol Lett. 2014;36(2):327-35.
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Part No. : BBa_K2230003

Type: Composite part

Cloning:

The fragment of RBS-EmR of RBS-EmR/pSB1C3 (BBa_K2230002) cut by EcoRI & SpeI was gel eluted and assembled with TT/pSB1C3 (Double terminator, BBa_B0015) cut by EcoRI & XbaI.

Part No. : BBa_K2230004

Type: Composite part (device)

Cloning:

RBS-EmR which was cut and gel-eluted from RBS-EmR/pSB1C3 (BBa_K2230002) was assembled with CP29-RBS-aeBlue/pLBA169 (BBa_K2230000)

Function:

A vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA, which encodes a surface layer protein A. EmR (erythromycin resistance gene), which is driven by the high constitutive promoter of slpA, acts as a selection marker. And aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will be erythromycin resistant and express blue color proteins.

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Background

Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.

PI promoter [BBa_K861170] is a modified version to Pcar with correction of -10 position in the promoter region, which gives the promoter stronger activity and a weaker CRP interaction. The PI promoter [BBa_K861170] was used as a major part in the work of team NCKU_Tainan in 2016. These two parts have been used in our work.

PhlF repressor system contains the repressor PhlF [BBa_K1725041] and the PhlF repressible promoter [BBa_K1725001] created by Glasgow in 2015. PhlF could repress GFP fluorescence intensity by 83-fold according to the study of Glasgow’s work.

We’ve innovated this year a novel glucose responsive repressor system (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 [BBa_K2230012]) by connecting these two system and extend the function of them. Furthermore, based on this new system, we assembled lysis and nuclease genes to the device and created the suicide circuit controlled by the presence of glucose (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP-sRBS-lysis-sRBS-NucA/pSB1C3 [BBa_K2230017]).

Lysis gene [BBa_K117000] created by NTU-Singapore in 2008 encodes Lysis protein which could not only lyse bacterial cell membrane but also activate the endonuclease of Colicin E7 (ColE7). The lysis-colicin is one class of bacteriocins which are produced to response to worsening environmental conditions and outcompete other bacteria1.

NucA [BBa_K1159105] created by TU-Munich in 2013 from Staphylococcus aureus produces a thermostable exo- and endo-nuclease that is able to degrade genomic DNAs2. NucA also has a role in the cleavage of extracellular DNAs and preventing biofilm formation.

The Parts

Part No. : BBa_K2230005

Type: Composite part (device)

Cloning:

PI-RBS-RFP was amplified from RFP Coding Device (BBa_J04450) using a primer with PI-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.

Function:

RFP can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, PI, is glucose responsive.

Part No. : BBa_K2230006

Type: Composite part (device)

Cloning:

Pcar-RBS-RFP was amplified from RFP Coding Device (BBa_J04450) using a primer with Pcar-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.

Function:

RFP can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.

Part No. : BBa_K2230007

Type: Composite part (device)

Cloning:

PI-RBS-aeBlue was amplified from CP29-RBS-aeBlue (BBa_K1033280) using a primer with PI-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.

Function:

aeBlue can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, PI, is glucose responsive.

Part No. : BBa_K2230008

Type: Composite part (device)

Cloning:

Pcar-RBS-aeBlue was amplified from CP29-RBS-aeBlue (BBa_K1033280) using a primer with Pcar-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.

Function:

aeBlue can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.

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Part No. : BBa_K2230009

Type: Composite part (device)

Cloning:

PI-RBS-PhlF-T was amplified from RBS + PhlF repressor + terminator (BBa_K1725041) using a primer with PI-RBS (B0032) sequence. The PCR product was ligated to pSB1C3 cut by XbaI and PstI.

Function:

Repressor PhlF can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, PI, is glucose responsive.

Part No. : BBa_K22300010

Type: Composite part (device)

Cloning:

Pcar-RBS-PhlF was amplified from RBS + PhlF repressor + terminator (BBa_K1725041) using a primer with Pcar-RBS (B0032) sequence. The PCR product was ligated to pSB1C3 cut by XbaI and PstI.

Function:

Repressor PhlF can be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, Pcar, is glucose responsive.

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Part No. : BBa_K2230011

Type: Composite part (device)

Cloning:

Pr-sRBS-GFP (BBa_K1725001) with strong RBS (B0034) was assembled with PI-RBS-PhlF-T/pSB1C3 (BBa_K2230009).

Function:

The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.

Part No. : BBa_K2230012

Type: Composite part (device)

Cloning:

Pr-sRBS-GFP (BBa_K1725001) with strong RBS (B0034) was assembled with Pcar-RBS-PhlF-T/pSB1C3 (BBa_K2230010)

Function:

The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.

Part No. : BBa_K2230013

Type: Composite part (device)

Cloning:

Pr-wRBS-GFP (BBa_K1725002) with weak RBS (B0032) was assembled with PI-RBS-PhlF-T/pSB1C3 (BBa_K2230009)

Function:

The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.

Part No. : BBa_K2230014

Type: Composite part (device)

Cloning:

Pr-wRBS-GFP (BBa_K1725002) with strong RBS (B0034) was assembled with Pcar-RBS-PhlF-T/pSB1C3 (BBa_K2230010)

Function:

The expression of GFP driven by PhlF-repressed promoter (named Pr here) was dose-dependently regulated by PhlF repressor and reduced upon an increasing concentrations of glucose. In other words, the intensity of GFP increased when glucose gradually ran out.

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Part No. : BBa_K2230015

Type: Composite part (device)

Cloning:

wRBS-lysis was amplified from Lysis gene (BBa_K117000) using a primer with weak RBS sequence (B0032) and assembled with Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 (BBa_K2230012)

Function:

Suicide genes of lysis was driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.

Part No. : BBa_K2230016

Type: Composite part (device)

Cloning:

sRBS-lysis was amplified from Lysis gene (BBa_K117000) using a primer with strong RBS sequence (B0034) and assembled with Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 (BBa_K2230012)

Function:

Suicide genes of lysis was driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.

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Part No. : BBa_K2230017

Type: Composite part (device)

Cloning:

sRBS-NucA was amplified from Mature Nuclease NucA from Staphylococcus aureus (BBa_K1159105) using a primer with strong RBS sequence (B0034) and assembled with Pcar-RSB-PhlF-T-Pr-sRBS-GFP-sRBS-lysis/pSB1C3

Function:

Suicide genes of lysis and nuclease (NucA) were driven under the PhlF-repressed promoter (Pr). And the expression of the repressor (PhlF) would be induced upon glucose by a glucose responsive promoter (Pcar). That is, the bacteria can be killed by the suicide genes in the absence of (or running out of) glucose.

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Experiment and Results



The result shown in Fig. 2 indicated that PI promoter has significant activity in LB culture media. However, the activity of Pcar promoter is greater than negative control but much smaller than PI and positive control. It’s consistent with the properties of PI and Pcar promoters just mentioned previously in GENE CLONING section and described previously in Part Resgistry [Part: BBa_K861170] by team WHU-China in 2012 who designed the promoters.

In our experiment as presented in Fig. 3, PI and Pcar promoters just responded to various concentrations of glucose with a very slight dose-dependent increase. This phenomenon didn’t correspond to the data provided by team WHU-China in 2012 and team NCKU_Tainan in 2016. Maybe our measurement was not in an optimized condition. Or the reporter of RFP activity was not sensitive enough to respond this difference.

The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD600 was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. 100 μl of the bacterial culture was put into one well of a black-walled, clear-bottom 96-well microplates (Thermo Fisher Scientific Inc.). The fluorescence intensity was measured using BioTek Synergy H1 Hybrid Multi-Mode Reader System at Ex/Em = 488nm/518nm for GFP.

In the assay for repressor system, the data in Fig. 2 gave the similar results as team Glasgow did in 2015, in which the strong activity of the repressible promoter was significantly repressed in the presence of PhlF repressor.

In the assay for glucose responsive repressor system, we improved the Glasgow’s BioBrick device, in which the expression of PhlF repressor was driven by a glucose response promoter, Pcar. The result in Fig 3 clearly indicated that the GFP activity driven by the repressible promoter was gradually increased in response to the loss of glucose to 1.88 folds compared to the initial GFP activity at the beginning culture in M9 media, suggesting that the level of expression of PhlF was positively corresponding to the concentration of glucose.



The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD600 was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. 100 μl of the bacterial culture was put into one well of a black-walled, clear-bottom 96-well microplates (Thermo Fisher Scientific Inc.). OD600 was measured using BioTek Synergy H1 Hybrid Multi-Mode Reader System. Further, the culture media were taken out and diluted 106 times following by spreading onto LB Cm agar plate at 37°C overnight. The third day, the numbers of colonies were counted and bacterial viability was calculated.

As you can see in Fig 7., the OD value in response to the decreasing concentration of glucose was gradually reduced to 1.89 much less than average 2.71 in control group without suicide gene expression, implying that the suicide proteins killed the cells in the loss of glucose in the environment. Moreover, when the bacteria were grown in M9 media with 0.5mM glucose for 4 hours, the survival rate was decreased to 34% compared to 56% of bacteria without suicide genes (Fig. 8). And the cell numbers were reduced to 671 compared to 1120 of bacteria without suicide genes. Both data confirmed that this suicide device works well and indicated that killing process began when glucose in the media was running out.

Background

Salmonella typhimurium LT2 has two glucose-specific transporter systems, PTS system and sodium/glucose cotransporter. PTS system contains two subunits IIA encoded by crr and IIBC by ptsG which are assembled to a high-affinity active transporter. The other is a Na+/glucose cotransporter encoded by STM1128 that contributes to facilitated transport with lower glucose affinity. Based on our research, the glucose transporter of Salmonella has a lower Km compared to human small intestine, Staphylococcus and E. coli (Table 1), indicating a higher efficiency for glucose uptake. In our project, we created the glucose transporter device and genetically engineer microbes with these two systems.

In order to express the genes in E. coli for demonstration and in probiotics for proof-of-concept in a real world. We chose promoter CP29 that is a strong constitutive promoter working well in both E. coli and Lactobacillus spp1. The biobrick part, CP29-RBS-aeBlue (BBa_K1033280) was used and to be assembled with the transporter genes.

Part No.: BBa_K2230018

Type: Basic Part

Cloning:

The crr gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3

Part No.: BBa_K2230019

Type: Composite Part

Cloning:

The crr gene was amplified from gDNA of Salmonella typhimurium using a primer with RBS sequence (B0034), and then cloned onto pSB1C3

Part No.: BBa_K2230020

Type: Basic Part

Cloning:

The ptsG gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3

Part No.: BBa_K2230021

Type: Composite Part

Cloning:

The ptsG gene was amplified from gDNA of Salmonella typhimurium using a primer with RBS sequence (B0034), and then cloned onto pSB1C3

Part No.: BBa_K2230022

Type: Basic Part

Cloning:

The STM1128 gene was amplified from gDNA of Salmonella typhimurium and cloned onto pSB1C3

Part No.: BBa_K2230023

Type: Composite Part

Cloning:

The STM1128 gene was amplified from gDNA of Salmonella typhimurium using a primer with RBS sequence (B0034), and then cloned onto pSB1C3

Part No.: BBa_K2230024

Type: Composite Part

Cloning:

The RBS-ptsG part (BBa_K2230022) was assembled with Double Terminator/pSB1C3 (BBa_B0015)

Part No.: BBa_K2230025

Type: Composite Part

Cloning:

The RBS-STM1128 part (BBa_K2230024) was assembled with Double Terminator/pSB1C3 (BBa_B0015)

Part No.: BBa_K2230026

Type: Composite Part

Cloning:

The RBS-crr part (BBa_K2230019) was assembled with RBS-ptsG-TT/pSB1C3 (BBa_K2230024)

Part No.: BBa_K2230027

Type: Composite Part (device)

Cloning:

Promoter CP29-RBS-aeBlue (BBa_K1033280) was assembled with RBS-crr-RBS-ptsG-TT/pSB1C3 (BBa_K2230026)

Function:

An active high-affinity glucose transporter system. Promoter CP29 is a constitutive and strong promoter which works well in both E. coli and Lactobacillus spp. The blue protein encoded by aeBlue gene acts as indicator for the gene expression in the transgenic strain. This device can help E. coli and Lactobacillus spp. efficiently take up and retrieve glucose in the environment.

Part No.: BBa_K2230028

Type: Composite Part (device)

Cloning:

Promoter CP29-RBS-aeBlue (BBa_K1033280) was assembled with RBS-STM1128-TT/pSB1C3 (BBa_K2230025)

Function:

A facilitated low-affinity glucose transporter system. Promoter CP29 is a constitutive and strong promoter which works well in both E. coli and Lactobacillus spp. The blue protein encoded by aeBlue gene acts as indicator for the gene expression in the transgenic strain. This device can help E. coli and Lactobacillus spp. efficiently take up and retrieve glucose in the environment.

Experiment and Results

To measure glucose uptake by the engineered E. coli expressing PTS system or Na+/glucose cotransporter, the bacteria were culture in LB broth supplemented with 34μg/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD600 = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.



Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.

Result

As shown in Fig. 3, the growth of E. coli expressing the PTS reporter (i.e., crr and ptsG genes) was seriously retardant. As I mentioned earlier, the E. coli overexpressing glucose transporter may lose viability because of the toxic metabolites produced in glycolytic pathway. Not surprisingly, the PTS overexpression bacteria was almost unable to absorb glucose.

Fig 3. The cell growth overnight and glucose uptake of E. coli expressing the PTS transporter in 4 hours

Fig. 4 represented that the cell growth of E. coli expressing the Na+/Glucose transporter was comparable and even slightly higher than the control group. The glucose began to be absorbed at the 3rd hour. The glucose uptake efficiency was achieved upto 97% in Na+/Glu group and greater than in control group with 1.2 times difference.

Fig 4. The cell growth overnight and glucose uptake of E. coli expressing the Na+/Glu transporter at different time point
Discussion

We’ve successfully genetically engineered E. coli expressing high-affinity active PTS transporter system and low-affinity facilitated Na+/glucose cotransporter system by screening in MacConkey and glycerol agar plate with chloramphenicol, respectively. Our data is consistent with the previous study that overexpressing glucose transporter genes do really harm E. coli partly due to disturbing glycolytic pathways.

PTS expressing bacteria grew difficultly in LB broth but Na+/glucose transporter expressing bacteria grew comparably with general E. coli, suggesting that high-affinity active transporter did interact with sugar-related substances in LB culture media and harm the bacterial survival.

In our results, glucose absorption by Na+/glucose transporter expressing bacteria was achieved to 97% after 4 hours with 1.2 times enhanced efficiency compared to the normal E. coli. To further increase the rate of glucose uptake, one may think about the glucose metabolism or conversion to other materials when entering into the cell.

  • - REFERENCE -
  • 1.     Two mechanisms for growth inhibition by elevated transport of sugar phosphates in Escherichia coli. J Gen Microbiol. 1992;138(10):2007-14.
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