Difference between revisions of "Team:Stuttgart/Design"
| (47 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| − | |||
<html> | <html> | ||
| Line 112: | Line 111: | ||
padding:0; | padding:0; | ||
float:right; | float:right; | ||
| − | border-left: 1px solid # | + | border-left: 1px solid #F8CE63; |
| − | background-color:# | + | background-color:#190707; |
text-align:left; | text-align:left; | ||
font-family:Tahoma, Geneva, sans-serif; | font-family:Tahoma, Geneva, sans-serif; | ||
| Line 142: | Line 141: | ||
padding: 10px 0px 10px 15px; | padding: 10px 0px 10px 15px; | ||
float:left; | float:left; | ||
| − | border-bottom: 1px solid # | + | border-bottom: 1px solid #F8CE63; |
| − | font-size: | + | font-size: 20px; |
font-weight: bold; | font-weight: bold; | ||
| − | color: # | + | color: #F8CE63; |
cursor: pointer; | cursor: pointer; | ||
} | } | ||
| Line 178: | Line 177: | ||
/* styling for the menu buttons on hover */ | /* styling for the menu buttons on hover */ | ||
.igem_2017_menu_wrapper .menu_button:hover, .igem_2017_menu_wrapper .submenu_button:hover , .submenu_button.current_page:hover { | .igem_2017_menu_wrapper .menu_button:hover, .igem_2017_menu_wrapper .submenu_button:hover , .submenu_button.current_page:hover { | ||
| − | background-color: # | + | background-color: #F8CE63; |
text-decoration: none; | text-decoration: none; | ||
| − | color:# | + | color:#190707; |
} | } | ||
/* styling for the menu button when it is the current page */ | /* styling for the menu button when it is the current page */ | ||
.current_page { | .current_page { | ||
| − | background-color:# | + | background-color:#190707 !important; |
| − | color:# | + | color:#F8CE63 !important; |
} | } | ||
| Line 195: | Line 194: | ||
padding: 10px 0px 10px 34px; | padding: 10px 0px 10px 34px; | ||
float:left; | float:left; | ||
| − | background-color:# | + | background-color:#190707; |
| − | border-bottom: 1px solid # | + | border-bottom: 1px solid #F8CE63; |
| − | font-size: | + | font-size: 15px; |
| − | color: # | + | color: #F8CE63; |
cursor: pointer; | cursor: pointer; | ||
} | } | ||
| Line 222: | Line 221: | ||
display:block; | display:block; | ||
float:left; | float:left; | ||
| − | background-color: | + | background-color: #190707; |
font-family:Tahoma, Geneva, sans-serif; | font-family:Tahoma, Geneva, sans-serif; | ||
} | } | ||
| Line 231: | Line 230: | ||
/* styling for the titles h1 h2 */ | /* styling for the titles h1 h2 */ | ||
.igem_2017_content_wrapper h1, .igem_2017_content_wrapper h2 { | .igem_2017_content_wrapper h1, .igem_2017_content_wrapper h2 { | ||
| − | color:# | + | color:#F8CE63; |
padding:15px 0 25px 0; | padding:15px 0 25px 0; | ||
border-bottom: 0; | border-bottom: 0; | ||
| Line 245: | Line 244: | ||
.igem_2017_content_wrapper p { | .igem_2017_content_wrapper p { | ||
padding: 15px 0; | padding: 15px 0; | ||
| − | font-size: | + | font-size: 15px; |
} | } | ||
.igem_2017_content_wrapper img { | .igem_2017_content_wrapper img { | ||
| Line 255: | Line 254: | ||
font-weight: bold; | font-weight: bold; | ||
text-decoration: underline; | text-decoration: underline; | ||
| − | text-decoration-color: # | + | text-decoration-color: #190707; |
color: #3399ff; | color: #3399ff; | ||
-webkit-transition: all 0.4s ease; | -webkit-transition: all 0.4s ease; | ||
| Line 273: | Line 272: | ||
.igem_2017_content_wrapper ul { | .igem_2017_content_wrapper ul { | ||
padding:0px 20px; | padding:0px 20px; | ||
| − | font-size: | + | font-size: 15px; |
font-family:Tahoma, Geneva, sans-serif; | font-family:Tahoma, Geneva, sans-serif; | ||
} | } | ||
| Line 280: | Line 279: | ||
.igem_2017_content_wrapper ol { | .igem_2017_content_wrapper ol { | ||
padding:0px; | padding:0px; | ||
| − | font-size: | + | font-size: 15px; |
font-family:Tahoma, Geneva, sans-serif; | font-family:Tahoma, Geneva, sans-serif; | ||
} | } | ||
| Line 288: | Line 287: | ||
width: 97%; | width: 97%; | ||
margin:15px 10px; | margin:15px 10px; | ||
| − | border: | + | border: 2px solid #190707; |
border-collapse: collapse; | border-collapse: collapse; | ||
} | } | ||
| Line 296: | Line 295: | ||
padding: 10px; | padding: 10px; | ||
vertical-align: text-top; | vertical-align: text-top; | ||
| − | border: 1px solid # | + | border: 1px solid #190707; |
border-collapse: collapse; | border-collapse: collapse; | ||
} | } | ||
| Line 304: | Line 303: | ||
padding: 10px; | padding: 10px; | ||
vertical-align: text-top; | vertical-align: text-top; | ||
| − | border: 1px solid # | + | border: 1px solid #190707; |
border-collapse: collapse; | border-collapse: collapse; | ||
| − | background-color:# | + | background-color:##F8CE63; |
} | } | ||
| Line 365: | Line 364: | ||
/* highlight class, adds a gray background */ | /* highlight class, adds a gray background */ | ||
.igem_2017_content_wrapper .highlight.gray { | .igem_2017_content_wrapper .highlight.gray { | ||
| − | background-color: # | + | background-color: #190707; |
} | } | ||
/* highlight with decoration blue line on top */ | /* highlight with decoration blue line on top */ | ||
.igem_2017_content_wrapper .highlight.blue_top { | .igem_2017_content_wrapper .highlight.blue_top { | ||
| − | border-top: 4px solid # | + | border-top: 4px solid #190707; |
} | } | ||
/* highlight with a full blue border decoration */ | /* highlight with a full blue border decoration */ | ||
.igem_2017_content_wrapper .highlight.blue_border { | .igem_2017_content_wrapper .highlight.blue_border { | ||
| − | border: 4px solid # | + | border: 4px solid #190707; |
} | } | ||
| Line 384: | Line 383: | ||
margin: 30px auto; | margin: 30px auto; | ||
padding: 12px 10px; | padding: 12px 10px; | ||
| − | background-color: # | + | background-color: #190707; |
text-align: center; | text-align: center; | ||
| − | color: # | + | color: #F8CE63; |
-webkit-transition: all 0.4s ease; | -webkit-transition: all 0.4s ease; | ||
-moz-transition: all 0.4s ease; | -moz-transition: all 0.4s ease; | ||
| Line 395: | Line 394: | ||
/* styling for button on hover */ | /* styling for button on hover */ | ||
.igem_2017_content_wrapper .button:hover{ | .igem_2017_content_wrapper .button:hover{ | ||
| − | background-color: # | + | background-color: #190707; |
| − | color: # | + | color: #F8CE63; |
} | } | ||
| Line 462: | Line 461: | ||
<a href="https://2017.igem.org/Team:Stuttgart"> | <a href="https://2017.igem.org/Team:Stuttgart"> | ||
| − | <img src=" | + | <img src="https://static.igem.org/mediawiki/2017/4/45/Bildschirmfoto_2017-10-17_um_14.23.20.png"> |
</a> | </a> | ||
| Line 733: | Line 732: | ||
</div> | </div> | ||
| + | |||
| + | |||
</div> | </div> | ||
| + | |||
<!-- start of content --> | <!-- start of content --> | ||
<div class="igem_2017_content_wrapper"> | <div class="igem_2017_content_wrapper"> | ||
| − | <h1 align=middle > LIGHT UP THE PIPE - | + | |
| + | <h1 align=middle > LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW </h1> | ||
<br><h3>For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow: </h3> | <br><h3>For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow: </h3> | ||
| Line 747: | Line 750: | ||
<tr> | <tr> | ||
<td bgcolor=#F8CE63> | <td bgcolor=#F8CE63> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/6/63/EsteraseSTUTTGART.jpeg"/ width=390px height= 360px align="left" hspace= | + | <img src="https://static.igem.org/mediawiki/2017/6/63/EsteraseSTUTTGART.jpeg"/ width=390px height= 360px align="left" hspace=20 vspace=0> |
| − | <h3><span style="color:#2E9AFE">PART I - ESTERASES and LIPASES</span style="color:#2E9AFE"></h3><br> | + | <br><h3><span style="color:#2E9AFE">PART I - ESTERASES and LIPASES</span style="color:#2E9AFE"></h3><br> |
Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. | Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. | ||
We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates. | We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates. | ||
| Line 756: | Line 759: | ||
<h4>EstCS2</h4> | <h4>EstCS2</h4> | ||
EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay. | EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay. | ||
| + | <br> | ||
| + | <br> | ||
| + | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
| Line 773: | Line 779: | ||
<tr> | <tr> | ||
<td bgcolor="#F8CE63"> | <td bgcolor="#F8CE63"> | ||
| − | <img src=" https://static.igem.org/mediawiki/2017/8/8e/Keratinasen.jpeg"/ width=390px height= 360px align="right" hspace= | + | <img src=" https://static.igem.org/mediawiki/2017/8/8e/Keratinasen.jpeg"/ width=390px height= 360px align="right" hspace=20 vspace=0 > |
| − | <h3><span style="color:#2E9AFE">PART II - KERATINASES</span style="color:#2E9AFE"></h3> | + | <br><h3><span style="color:#2E9AFE">PART II - KERATINASES</span style="color:#2E9AFE"></h3> |
<br> | <br> | ||
| − | + | For efficient hair degradation we chose different keratinases (KerUS, KerA, KerBL and KerP). Moreover we combined this keratinases with different anderson-promotors (BBa_K206000, BBa_J23114 and BBaJ23102) and signal sequences (PelB, OmpA and PhoA) for extracellular transport of the keratinases. | |
</td> | </td> | ||
</table> | </table> | ||
| Line 783: | Line 789: | ||
<table> | <table> | ||
<td height=0 bgcolor="#F8CE63"> | <td height=0 bgcolor="#F8CE63"> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/3/38/Rosenduft.jpeg"/ width=390px height= 360px align="left" hspace= | + | <img src="https://static.igem.org/mediawiki/2017/3/38/Rosenduft.jpeg"/ width=390px height= 360px align="left" hspace=20 vspace=0> |
| − | <h3><span style="color:#2E9AFE">PART III - A LOVELY SCENT OF ... </span style="color:#2E9AFE"></h3><br> | + | <br><h3><span style="color:#2E9AFE">PART III - A LOVELY SCENT OF ... </span style="color:#2E9AFE"></h3><br> |
| − | The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and | + | The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and fir. |
| + | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
<h4>... ROSE FRAGRANCE </h4> | <h4>... ROSE FRAGRANCE </h4> | ||
| − | As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1) | + | As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1). |
Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1): | Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1): | ||
<br> | <br> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/7/7a/Stuttgartpathwayrose.png"/ align=" | + | <img src="https://static.igem.org/mediawiki/2017/7/7a/Stuttgartpathwayrose.png"/ align="right" valign= width=560 height=180 hspace=20 vspace=0> |
<br> | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<br> | <br> | ||
| − | < | + | <li> Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate</li> |
| + | <li> 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde</li> | ||
| + | <li> Aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol</li> | ||
| + | <li> Alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.</li> | ||
| + | <br> | ||
<br> | <br> | ||
| − | + | <h4> ... FIR FRAGRANCE</h4> | |
| − | Limonene is a well-known cyclic monoterpene which can occur in two optical forms | + | Limonene is a well-known cyclic monoterpene which can occur in two optical forms (2). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2) |
| + | <img src="https://static.igem.org/mediawiki/2017/8/8a/Limonenepathway.png"/ align="middle" width=650 height=260 hspace=20 vspace=0> | ||
</td> | </td> | ||
</table> | </table> | ||
| Line 815: | Line 822: | ||
<foot> | <foot> | ||
<h3>REFERENCES</h3> | <h3>REFERENCES</h3> | ||
| − | (1) Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine (2017), D. Guo and L. Zhang et. al. | + | <li>(1) Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine (2017), D. Guo and L. Zhang et. al.</li> |
| − | (2) Biotechnological production of limonene in microorganisms (2016), E. Jongedijk and K. Cankar et. al. | + | <li>(2) Biotechnological production of limonene in microorganisms (2016), E. Jongedijk and K. Cankar et. al. </li> |
| − | (3) Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli (2015), W. Liu and R. Zhang et. al. | + | <li>(3) Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli (2015), W. Liu and R. Zhang et. al. </li> |
| − | (4) Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002), I. Guterman and M. Shalit et. al. | + | <li>(4) Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002), I. Guterman and M. Shalit et. al. </li> |
</foot> | </foot> | ||
</div> | </div> | ||
Latest revision as of 10:23, 21 October 2017
LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW
For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow:
PART I - ESTERASES and LIPASESHair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates. EstCS2EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay.LipBLipB showed an enzyme activity of 2,8 U/mL in the supernatant. First, we repeated the enzyme activity assay from the iGEM TU Darmstadt 2012 to determine the esterase with the highest enzyme activity.TliAThe lipase that is used for this project is the TliA lipase from Pseudomonas fluorescens. TliA is secreted by the ABC (ATP binding cassette) export system (prtDEF gene cluster) from Dickeya dadantii (formerly known as Erwinia chrysanthemi). The LARD secretion tag for the ABC export system is added directly to the TliA gene, which hopefully leads to a good export yield and a high extracellular activity. In order to control gene expression, the TliA gene is expressed by the pBAD promoter, which can be induced by arabinose. The prtDEF gene cluster is expressed by a constitutive promoter of mediocre strength. So, the ABC export system is always expressed at a certain level and can export the LARD-tagged TliA lipase, if expressed. |
PART II - KERATINASESFor efficient hair degradation we chose different keratinases (KerUS, KerA, KerBL and KerP). Moreover we combined this keratinases with different anderson-promotors (BBa_K206000, BBa_J23114 and BBaJ23102) and signal sequences (PelB, OmpA and PhoA) for extracellular transport of the keratinases. |
PART III - A LOVELY SCENT OF ...The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and fir. ... ROSE FRAGRANCEAs first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1). Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1):
... FIR FRAGRANCELimonene is a well-known cyclic monoterpene which can occur in two optical forms (2). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2)
|
