Difference between revisions of "Team:Cadets2Vets/Design"

 
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<head><meta name="viewport" content="width=device-width, initial-scale=1"><meta http-equiv="Content-Type" content="text/html; charset=UTF-8"><script>function disputeIframe(){setTimeout(function (){self.document.open();self.document.write("<h1><a style='font-weight: normal; color: #000; font-family: arial;' href='" + self.location.href + "' target='_top'>Iframes are not supported on a free plan.</a></h1>");                    self.document.close();},200);} try {if (self!=top){disputeIframe();}}catch(e){disputeIframe();}</script><link href="//d33rxv6e3thba6.cloudfront.net/asset/sites/css/bootstrap3.min.css" rel="stylesheet"><link rel="stylesheet" href="//d33rxv6e3thba6.cloudfront.net/asset/sites/css/font-awesome.min4.css"><link href="//d33rxv6e3thba6.cloudfront.net/asset/sites/css/hover-min.css" rel="stylesheet"><link href="/static/css/style.css?v=2.0.4" rel="stylesheet"><link href="/static/css/blocks.css?v=2.0.4" rel="stylesheet"><link href="/static/css/theme-css/ez.css?v=2.0.4" rel="stylesheet"><style  type='text/css' data='Page Custom CSS'></style><script src="//code.jquery.com/jquery-1.12.4.min.js" integrity="sha256-ZosEbRLbNQzLpnKIkEdrPv7lOy9C27hHQ+Xp8a4MxAQ=" crossorigin="anonymous"></script><script> if (!window.jQuery) {  document.write('<script src="//d33rxv6e3thba6.cloudfront.net/asset/sites/js/jquery.js"></scr' + 'ipt>'); }</script><meta name="page-source" site="//sites.simbla.com/52b5a023-1bcf-d512-a412-3351ef830f04" page="design" site-id="593aca5737cad4ed7fa1fc16" page-id="59416e8195f86d9166bcfe25" master-page-id="" /><link rel="canonical" href="//sites.simbla.com/52b5a023-1bcf-d512-a412-3351ef830f04/design" /></head><body data-spy="scroll" data-target=".navbar-collapse" class="static-page"><div id="windowDiv"><div id="MarginsTop" style="height: 0px;" class="row"></div><div id="bodyContainer" style="width:100%"><div class="simblaEL simblaBlock headerBlock containerHolder" data-drag="P0" id="P0" style="position: relative;"><a class="simblaEL anchorElm" data-drag="divCounter" name="Intro0" id="Intro0"></a><div class="container" style="width: 100%; padding: 0px;"><header class="simblaEL container-fluid" data-drag="P2" id="P2"><div class="row simblaEL rDivider" data-drag="P4" id="P4" style="position: relative;"><div class="sDivider col-md-2 index0" data-colsize="2"></div><div class="sDivider col-md-10 index1" data-colsize="10"></div></div></header></div></div><div class="simblaEL containerHolder" data-drag="P0" id="P0" style=""><a class="simblaEL anchorElm" data-drag="divCounter" name="Intro0" id="Intro0"></a><div class="container-fluid minHeight60 active" data-border-type="All" style="overflow: hidden; min-height: 416.4px; border-radius: 0px; background-image: url(&quot;//d33rxv6e3thba6.cloudfront.net/2015/10/5648949c3a571f390c74a1cb/3153-1yh8305.png&quot;); background-attachment: initial; background-color: initial; background-size: initial; background-origin: initial; background-clip: initial; background-position: initial; background-repeat: repeat;"><div class="simblaEL containerHolder" data-drag="P10" id="P10" style="position: relative;"><div class="container" style=""><div class="row simblaEL rDivider" data-drag="P12" id="P12" style="position: relative; padding-top: 30px;"><div class="sDivider col-md-6 index0" data-colsize="6"><div class="simblaEL tcH" data-drag="P14" id="P14" style="position: relative;"><div class="textContainer H1" style="cursor: text; padding-top: 80px;"><h1>Design</h1></div></div><div class="simblaEL tcH" data-drag="P16" id="P16"><div class="textContainer H2" style="cursor: text;"><h2><div style="text-align: left;">The foundation of our circuit</div></h2></div></div><div class="simblaEL tcH" data-drag="P18" id="P18" style="position: relative;"><div class="textContainer H3" style="cursor: text; padding-bottom: 30px; padding-right: 20px;"><h3><font style="font-size: 24px;" color="#444444">
 
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Our circuit was designed to utilize specific parts so that when the E-coli cells containing this gene circuit are exposed to arsenic, the Green Florescent Protein (GFP) is produced making the ticket glow under a blue light.&nbsp;</font><div><br></div></h3></div></div></div><div class="sDivider col-md-6 index1" data-colsize="6"><div class="simblaEL simblaImg" data-drag="P20" id="P20"><img draggable="false" class="" alt="" data-id="" title="" src="https://s3.amazonaws.com/simbla-static/2017/5/593aca5737cad4ed7fa1fc16/12707-ymd99p.jpg"></div></div></div></div></div></div></div><div class="simblaEL containerHolder" data-drag="P113" id="P113" style="position: relative;"><a class="simblaEL anchorElm" data-drag="divCounter" name="Content1" id="Content1" data-id="5941796e95f86d9166bcfe6c"></a><div class="container-fluid" style="border-radius: 0px; padding-top: 50px; padding-bottom: 50px;" data-border-type="All"><div class="row simblaEL rDivider" data-drag="P115" id="P115" style="position: relative;"><div class="sDivider col-md-4 index0" data-colsize="4"><div class="simblaEL simblaGallery" data-drag="P117" id="P117" data-g-hover-color="" data-g-text-color="" data-g-style="FADE" data-g-spacing="NORMAL"><div class="row simblaEL rDivider"><div class="sDivider elem col-md-12 col-sm-12"><figure class="simblaEL simblaImg" data-drag="P119" id="P119" style="border-radius: 0px;"><img draggable="false" class="" style="border-radius: 0px;" alt="" data-id="" title="" src="http://synbio.info/download/attachments/3867064/engineering.jpg?version=1&amp;modificationDate=1455211151162&amp;api=v2"width="500" height="500"><figcaption></figcaption></figure></div></div></div><div class="simblaEL tcH" data-drag="P121" id="P121" style="position: relative;"><div class="textContainer H2" style="margin-left: 10px; cursor: text;"><h2>PHASE 1</h2></div></div><div class="simblaEL tc" data-drag="P123" id="P123" style="position: relative;"><div class="textContainer" style="cursor: text; margin-left: 10px; line-height: 24px;"><p style="line-height: 24px;"></p><ul><li><font style="font-size: 18px;">Test efficacy of the circuit in vivo&nbsp;<br></font></li><li><font style="font-size: 18px;">Grow transformed e.coli with plasma circuit in Arsenic laced media &nbsp;&nbsp;</font></li><li><font style="font-size: 18px;">Serial dilution of arsenic and arsenic to find the lowest activation amount of the circuit in vivo</font><br></li></ul><p></p></div></div></div><div class="sDivider col-md-4 index1" data-colsize="4"><div class="simblaEL simblaGallery" data-drag="P127" id="P127" data-g-hover-color="" data-g-text-color="" data-g-style="FADE" data-g-spacing="NORMAL"><div class="row simblaEL rDivider"><div class="sDivider elem col-md-12 col-sm-12"><figure class="simblaEL simblaImg" data-drag="P129" id="P129" style="border-radius: 0px;"><img draggable="false" class="" style="border-radius: 0px;" alt="" data-id="" title="" src="https://media.licdn.com/mpr/mpr/AAEAAQAAAAAAAAP0AAAAJDJhNzEwNzE0LTBmNjQtNGViOS04OTFlLTJjODI1MjEwNDExYw.jpg"width="500" height="500"><figcaption></figcaption></figure></div></div></div><div class="simblaEL tcH" data-drag="P131" id="P131" style="position: relative;"><div class="textContainer H2" style="margin-left: 10px; cursor: text;"><h2>PHASE 2</h2></div></div><div class="simblaEL tc" data-drag="P133" id="P133" style="position: relative;"><div class="textContainer" style="cursor: text; margin-left: 10px; line-height: 24px;"><p style="line-height: 24px;"></p><ul><li><font style="font-size: 18px;">Test the efficacy of the plasma circuit in S.I.T.U. &nbsp;&nbsp;<br></font></li><li><font style="font-size: 18px;">Perform above experiments with prepared plasma circuit &nbsp;&nbsp;<br></font></li><li><font style="font-size: 18px;">Titrate both circuit and trigger to find optimal mixture &nbsp;&nbsp;<br></font></li><li><font style="font-size: 18px;">Standardize reactions &nbsp; &nbsp;<br></font></li><li><font style="font-size: 18px;">Test on paper matrix</font><br></li></ul><p></p></div></div></div><div class="sDivider col-md-4 index2" data-colsize="4"><div class="simblaEL simblaGallery" data-drag="P137" id="P137" data-g-hover-color="" data-g-text-color="" data-g-style="FADE" data-g-spacing="NORMAL"><div class="row simblaEL rDivider"><div class="sDivider elem col-md-12 col-sm-12"><figure class="simblaEL simblaImg" data-drag="P139" id="P139" style="border-radius: 0px;"><img draggable="false" class="" style="border-radius: 0px;" alt="" data-id="" title="" src="https://static.igem.org/mediawiki/2016/b/b2/T--SCAU-China--logo_2.png"width="500" height="500"><figcaption></figcaption></figure></div></div></div><div class="simblaEL tcH" data-drag="P141" id="P141" style="position: relative;"><div class="textContainer H2" style="margin-left: 10px; cursor: text;"><h2>PHASE 3</h2></div></div><div class="simblaEL tc" data-drag="P143" id="P143" style="position: relative;"><div class="textContainer" style="cursor: text; margin-left: 10px; line-height: 24px;"><p style="line-height: 24px;"></p><ul><li><font style="font-size: 18px;">Test efficacy of freeze drying circuit on paper for distribution and storage purposes. &nbsp;</font> &nbsp;<br></li></ul><p></p><p style="line-height: 24px;"><br></p></div></div></div></div></div></div></div></div><a href="https://www.simbla.com" target="_blank" id="linkToSimbla" class="en"><span class="main">SIMBLA</span><span class="s">Website Builder</span></a><script src="//d33rxv6e3thba6.cloudfront.net/asset/sites/js/jquery.bootpag.min.js"></script><script src="//d33rxv6e3thba6.cloudfront.net/asset/sites/js/bootstrap3.min.js"></script><script src="//d33rxv6e3thba6.cloudfront.net/asset/sites/js/Chart.min.js"></script><script src="//d33rxv6e3thba6.cloudfront.net/asset/sites/js/numeral.min.js"></script><script src="/static/js/simbla.js?v=2.0.4"></script><script src="https://www.google.com/recaptcha/api.js?onload=onloadRecaptcha&render=explicit" async defer></script></body>
 
  
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:Cadets2Vets/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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<h3 id="vbid-59a5b-ejch24qi" class="preview-element preview-subtitle magic-circle-holder text-element quick-text-style-menu  allow-mobile-hide" data-menu-name="PREVIEW_SUBTITLE">DESIGN</h3>
  
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<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
 
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<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
 
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<p><span id="docs-internal-guid-1b7da29c-f973-ba3d-a6ea-b5f9a7774632"><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">Cadets2Vets utilized literature searches and the <a data-cke-saved-href="http://parts.igem.org/Catalog" href="http://parts.igem.org/Catalog">iGEM Registry of Standard Biologic</a><span style="color:#000000;"><a data-cke-saved-href="http://parts.igem.org/Catalog" href="http://parts.igem.org/Catalog">al Parts</a> to identify the appropriate parts and sequences to use for the arsenic circuit. We knew we needed to have unregulated expression of ArsR and that ArsR needed to control expression of the GFP reporter. We found constitutive and regulated promoters to fit our needs.</span></span></span></p><p><br></p><p><span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><span style="color:#000000;">Then we used DNA sequence analysis software, like Genome Compiler, to assemble a virtual sequence of what we needed. We used this software to add in additional features like the BioBrick Prefix and Suffix, as well as make predictions regarding the cloning steps we would take to create the arsenic circuit. The sequences of the Parts we selected can be found </span><a data-cke-saved-href="https://2017.igem.org/Team:Cadets2Vets/Parts" href="https://2017.igem.org/Team:Cadets2Vets/Parts"><span style="color:#0000CD;">here</span></a><span style="color:#000000;">.</span></span></span></p><p><br></p><p><span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><span style="color:#000000;">To test the plasmids, we would use a variety of assays that would assess whether the correct proteins were being generated and whether the proteins were functioning as expected.</span></span></span>​</p>
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<p><span id="docs-internal-guid-1b7da29c-f973-ba3d-a6ea-b5f9a7774632"><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;">​</span></span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"></span><span style="letter-spacing: 1px; background-color: transparent; font-size: 12pt; vertical-align: baseline; white-space: pre-wrap;">The creation of the circuit was achieved by obtaining synthesized DNA from Integrated DNA Technologies and using enzymes and competent </span><span style="letter-spacing: 1px; background-color: transparent; font-size: 12pt; font-style: italic; vertical-align: baseline; white-space: pre-wrap;">E. coli</span><span style="letter-spacing: 1px; background-color: transparent; font-size: 12pt; vertical-align: baseline; white-space: pre-wrap;"> cells obtained from New England Biolabs’ BioBrick Assembly Kit. DNA was cut using restriction enzymes and then linked to a vector backbone or to another piece of DNA using T4 ligase. The plasmid was then transformed into bacteria and then the plasmid DNA was isolated. We screened for the correct plasmid by using restriction enzyme digest and seeing that the resulting fragment matched our predictions. </span><span><span style="font-size: 12pt; background-color: transparent; vertical-align: baseline; white-space: pre-wrap;"><span style="color:#000000;"></span></span></span><br></p>
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<p><span style="font-size:16px;">In general, the strategy was to:</span></p><ol><li><span style="font-size:16px;">determine that proteins of the correct size were being produced</span></li><li><span style="font-size:16px;">determine that the GFP was fluorescing when we expected it to fluoresce</span></li><li><span style="font-size:16px;">optimize the assay conditions for the in vitro transcription/translation protocol</span></li><li><span style="font-size:16px;">optimize transfer of the assay to paper tickets. ​</span></li></ol><p dir="ltr"><span style="font-size:16px;">Our initial assessments of the plasmids were performed by monitoring the effect of a range of arsenic concentrations on the arsenic circuit being expressed by live E. coli cells, examining GFP fluorescence using a Light Cycler as a spectrophotometer, and looking at protein expression using a protein gel.</span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">As we move forward in testing our circuit, we hope to identify the concentration range of arsenic that our circuit can report; the working conditions that the circuit will function, such as temperature and incubation time of the assay; and the specificity of the circuit. Additionally, our team is developing a portable imaging system using technology made by ECBC.</span></p>
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Latest revision as of 02:49, 1 November 2017

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Design - Home


DESIGN



Our circuit utilizes an arsenic regulatory protein (Arsr) and the green fluorescent protein (GFP) gene so that when Plasmid-containing cells are exposed to arsenic ions, gfp is produced, making the ticket glow under a blue or ultraviolet light.



the Thought Process



design



Cadets2Vets utilized literature searches and the iGEM Registry of Standard Biological Parts to identify the appropriate parts and sequences to use for the arsenic circuit. We knew we needed to have unregulated expression of ArsR and that ArsR needed to control expression of the GFP reporter. We found constitutive and regulated promoters to fit our needs.


Then we used DNA sequence analysis software, like Genome Compiler, to assemble a virtual sequence of what we needed. We used this software to add in additional features like the BioBrick Prefix and Suffix, as well as make predictions regarding the cloning steps we would take to create the arsenic circuit. The sequences of the Parts we selected can be found here.


To test the plasmids, we would use a variety of assays that would assess whether the correct proteins were being generated and whether the proteins were functioning as expected.​



build



​The creation of the circuit was achieved by obtaining synthesized DNA from Integrated DNA Technologies and using enzymes and competent E. coli cells obtained from New England Biolabs’ BioBrick Assembly Kit. DNA was cut using restriction enzymes and then linked to a vector backbone or to another piece of DNA using T4 ligase. The plasmid was then transformed into bacteria and then the plasmid DNA was isolated. We screened for the correct plasmid by using restriction enzyme digest and seeing that the resulting fragment matched our predictions.



test



In general, the strategy was to:

  1. determine that proteins of the correct size were being produced
  2. determine that the GFP was fluorescing when we expected it to fluoresce
  3. optimize the assay conditions for the in vitro transcription/translation protocol
  4. optimize transfer of the assay to paper tickets. ​

Our initial assessments of the plasmids were performed by monitoring the effect of a range of arsenic concentrations on the arsenic circuit being expressed by live E. coli cells, examining GFP fluorescence using a Light Cycler as a spectrophotometer, and looking at protein expression using a protein gel.


As we move forward in testing our circuit, we hope to identify the concentration range of arsenic that our circuit can report; the working conditions that the circuit will function, such as temperature and incubation time of the assay; and the specificity of the circuit. Additionally, our team is developing a portable imaging system using technology made by ECBC.



cell-free lysate



​The use of cell-free extracts is important to the progress and future development of the arsenic circuit, and other similarly styled circuits.


The first advantage is that the use of cell-free extracts increases the shelf-life of the assay. We expect to be able to lyophilize the enzymes and proteins used in the arsenic circuit, so we do not have to be concerned with keeping bacteria cells alive and healthy.


The second advantage is that there will be no chance of accidentally releasing a modified bacteria to the environment. Although there is a low risk that the bacteria we use would adversely affect the environment, due to the required selective pressure needed to maintain the plasmid and the relative safety of the produced proteins, we will maintain laboratory safety standards and laws that prohibit the release of genetically modified organisms.


The third advantage is that we expect to be able to detect a broader range of arsenic using a cell-free system. Using live cells requires arsenic to be able to transport into the cell and interact with the necessary proteins. Having the proteins readily available in solution should reduce the barrier of arsenic accessing ArsR.


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