Difference between revisions of "Team:Cadets2Vets/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.

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.

In general, the strategy was to:

determine that proteins of the correct size were being produced
determine that the GFP was fluorescing when we expected it to fluoresce
optimize the assay conditions for the in vitro transcription/translation protocol
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.

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.

Difference between revisions of "Team:Cadets2Vets/Design"

Revision as of 23:43, 25 October 2017

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

build

test

cell-free lysate

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2304 Jefferson Ave, Tacoma, WA 98402

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-	<h3 id="vbid-ae64641e-er2wlbbf" class="preview-element preview-subtitle magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_SUBTITLE">DESIGN</h3>
+	<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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 <div class="preview-title-holder removable-parent order-handle">
-	<h1 id="vbid-ae64641e-igbt0u4c" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >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 U.V. light. <br></h1>
+	<h1 id="vbid-59a5b-dnp8mtfr" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >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. <br></h1>
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-		<p>THE FOUNDATION OF OUR CIRCUIT</p>
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 <div class="preview-title-holder removable-parent order-handle">
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 <div class="preview-title-holder removable-parent order-handle">
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+	<h2 id="element-287f1dcd2868fb6" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >design</h2>
 </div>
 <br class="lower-line-break" />
@@ Line 499: / Line 820: @@
 <br class="upper-line-break" />
-<div class="preview-divider-holder removable-parent order-handle">
+<div class="preview-body-holder removable-parent order-handle">
-	<div id="vbid-ae64641e-luiljz1l" class="preview-element preview-divider magic-circle-holder quick-text-style-menu" data-menu-name="PREVIEW_DIVIDER">
+	<div id="vbid-88f5fc9c-t14shpss" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
+		<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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 </div>
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 <div class="preview-title-holder removable-parent order-handle">
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+	<h2 id="vbid-34ff8b6b-wmxuvsaa" class="preview-element preview-title magic-circle-holder inner-page text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_TITLE" >build</h2>
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@@ Line 597: / Line 965: @@
 <br class="upper-line-break" />
-<div class="preview-divider-holder removable-parent order-handle">
+<div class="preview-body-holder removable-parent order-handle">
-	<div id="vbid-ae64641e-agt22xfb" class="preview-element preview-divider magic-circle-holder quick-text-style-menu" data-menu-name="PREVIEW_DIVIDER">
+	<div id="vbid-34ff8b6b-rhpe0gbn" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
+		<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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 <br class="upper-line-break" />
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-		<ul style="box-sizing: border-box; line-height: 1.5em; list-style-type: square; margin: 0.3em 0px 0px 1.6em; padding-top: 0px; padding-right: 20px; padding-bottom: 0px; list-style-image: url(&quot;https://2017.igem.org/wiki/skins/Igem/resources/bullet.gif?2015-09-09T00:46:40Z&quot;); font-size: 13px; font-family: Tahoma, Geneva, sans-serif; color: rgb(0, 0, 0); text-align: start;"><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFF0;"><font style="box-sizing: border-box; font-size: 18px;">Test efficacy of the circuit in vivo </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFF0;"><font style="box-sizing: border-box; font-size: 18px;">Grow transformed e.coli with plasma circuit in Arsenic laced media   </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFF0;"><font style="box-sizing: border-box; font-size: 18px;">Serial dilution of arsenic and arsenic to find the lowest activation amount of the circuit in vivo</font></span></li></ul>
+		<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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+	<div id="vbid-bd0b19d9-x6fcw58a" class="preview-element preview-body magic-circle-holder text-element quick-text-style-menu   allow-mobile-hide" data-menu-name="PREVIEW_BODY">
-		<ul style="box-sizing: border-box; line-height: 1.5em; list-style-type: square; margin: 0.3em 0px 0px 1.6em; padding-top: 0px; padding-right: 20px; padding-bottom: 0px; list-style-image: url(&quot;https://2017.igem.org/wiki/skins/Igem/resources/bullet.gif?2015-09-09T00:46:40Z&quot;); font-size: 13px; font-family: Tahoma, Geneva, sans-serif; color: rgb(0, 0, 0); text-align: start;"><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Test the efficacy of the plasma circuit in S.I.T.U. </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Perform above experiments with prepared plasmid circuit </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Titrate both circuit and trigger to find optimal mixture </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Standardize reactions </font></span></li><li style="box-sizing: border-box; margin-bottom: 0.1em;"><span style="color:#F0FFFF;"><font style="box-sizing: border-box; font-size: 18px;">Test on paper matrix</font></span></li></ul>
+		<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:16px;">The use of cell-free extracts is important to the progress and future development of the arsenic circuit, and other similarly styled circuits.</span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">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. </span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">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.</span></p><p dir="ltr"><br></p><p dir="ltr"><span style="font-size:16px;">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. </span></p>
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 		</div>
+		<!-- FOOTERS END -->
-			</div>
-		</div>
-	</div>
-</div>
-			</div>
-		</div>
-		<!-- PRICING END -->
@@ Line 1,102: / Line 1,453: @@
 	</div>
-	<script src="//xprs.imcreator.com/js/lightbox.js?v=1.4.1b"  type="text/javascript"></script>
+	<script type="text/javascript" src="https://2017.igem.org/Team:Cadets2Vets/js/lightbox?action=raw&ctype=text/javascript"></script>
-	<script src="//xprs.imcreator.com/js/spimeengine.js?v=1.4.1b"  type="text/javascript"></script>
+		<script type="text/javascript" src="https://2017.igem.org/Team:Cadets2Vets/js/spimeengine?action=raw&ctype=text/javascript"></script>
 	<!--  SHOPROCKET  ECOMMERCE  -->
@@ Line 1,115: / Line 1,465: @@
 </body>
-	<div id="user-pref" style="display:none;" data-lang="en"></div>
-	<div style="display:none;" id="lightbox-menus-holder">
-<div  class="login-dialog-wrapper white_content delay-anim" style="text-align:center;">
-	<div class="login dialog-holder">
-		<div  class="login-title"><span class="im-text form-type  t-t">Login</span></div>
-		<form id="login-form">
-			<img id="user-gravatar" src="https://www.gravatar.com/avatar/?d=mm" /><br/>
-			<input name="name" type="text" placeholder="nickname/email" data-placeholder="nickname/email" /><br/>
-			<input name="password" type="password" placeholder="password" data-placeholder="password" autocomplete="off" /><br/>
-			<input id="login-btn" class="clickable" type="button" class="input-btn" value="LOGIN">
-			<div id="goto-register" class="clickable t-t">Create a new account</div>
-			<div id="forgot-password" class="clickable t-t">Forgot Password?</div>
-		</form>
-	</div>
-	<div class="register dialog-holder" style="display:none;">
-		<div  class="login-title"><span class="im-text form-type  t-t">Login</span></div>
-		<form id="register-form" class="hide-phone">
-			<img id="user-gravatar" src="https://www.gravatar.com/avatar/?d=mm" /><br/>
-			<input name="email" type="text" placeholder="email" data-placeholder="email"  /><br/>
-			<input name="name" type="text" placeholder="nickname" data-placeholder="nickname" /><br/>
-			<input name="password" type="password"  placeholder="password" data-placeholder="password" autocomplete="off" /><br/>
-			<input id="terms" type="checkbox" checked="checked" disabled /><span class="form-label"><a href="http://www.imcreator.com/terms-of-service" target="_blank" class="terms-link  t-t">Terms and Conditions</a></span><br/>
-			<input id="register-btn" class="clickable" type="button" class="input-btn" value="JOIN">
-			<div id="goto-login" class="clickable t-t">Already a member?</div>
-		</form>
-	</div>
-	<div class="forgot-pass dialog-holder" style="display:none;">
-		<div  class="login-title"><span class="im-text form-type t-t">Login</span></div>
-		<form id="forgot-pass-form">
-			<img id="user-gravatar" src="https://www.gravatar.com/avatar/?d=mm" /><br/>
-			<input name="email" type="text" placeholder="email" data-placeholder="email" /><br/>
-			<input id="forgot-pass-btn" class="clickable" type="button" class="input-btn" value="SEND">
-			<div id="goto-login" class="clickable t-t">Back</div>
-		</form>
-	</div>
-</div>
-	</div>
-	<div id="fade" class="black_overlay fadein-noscript" style="display:none;"></div>
 </html>