Difference between revisions of "Team:Cornell/Composite Part"

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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://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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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://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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    <title>Team:Cornell/Composite - 2018.igem.org</title>
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                        <a href="https://2018.igem.org/Team:Cornell"><img src="https://static.igem.org/mediawiki/2018/a/af/T--Cornell--OscillateLogo.png" alt="Oscillate"></a>
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                                    <li class = "wet-lab-list-title"><b>WET LAB</b></li>
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                                    <li><a href="https://2018.igem.org/Team:Cornell/Foundations">FOUNDATIONS</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Cornell/Demonstrate">DEMONSTRATE</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Cornell/InterLab">INTERLAB</a></li>
 +
                                    <li><a href="https://2018.igem.org/Team:Cornell/Parts">PARTS</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Cornell/Basic_Part">BASIC PARTS</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Cornell/Composite_Part">COMPOSITE PARTS</a></li>
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                                    </li>
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                                </div>
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                                    <li class = "doc-list-title"><b>DOCUMENTATION</b></li>
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        </nav>
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        <!------------------------ NAV BAR END ------------------------>
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        <!------------------------ COMPOSITE PAGE BANNER START ------------------------>
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        <header class="no-pic-banner">         
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            <svg viewBox="0 0 100 100" width=100% height=100%>
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                <polygon points="-50,50 50,10 150,50 50,90" fill="white" fill-opacity="0.4"></polygon>
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                <text class="standard-page-banner-title" text-anchor="middle" alignment-baseline="middle" x=50% y=50% >Composite Part</text>
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        </header>
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        <!------------------------ COMPOSITE PAGE BANNER END ------------------------>
  
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        <!------------------------ STANDARD PAGE SIDE BAR + CONTENT START ------------------------>
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        <div class="standard-page-side-bar-content-wrapper">
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            <!------------------------ STANDARD PAGE SIDE BAR START ------------------------>
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            <div class="standard-page-side-bar-wrapper">
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                <ul class="standard-page-side-bar">
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                    <li><a href="#reporterconstructs">REPORTER CONSTRUCTS</a></li>
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                    <li><a href="#combinationconstructs">COMBINATION CONSTRUCTS</a></li>
 +
                </ul>
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            </div>
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            <!------------------------ STANDARD PAGE SIDE BAR END ------------------------>
  
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<div class="column full_size">
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<h1>Composite Parts</h1>
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            <div class="standard-page-content-wrapper">
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                <div class="standard-page-content-section">
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                    <p class="standard-page-content-text">
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<p>
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                        This year, we are proud to present a variety of parts that ultimately allow for control of gene expression using specific frequency-based signals. The final gene products of these constructs can be replaced by any gene of interest, making these parts a versatile platform for precisely regulating a gene’s expression in multi-plasmid or construct systems. We have submitted BBa_K2561003 for consideration for best composite part.
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A composite part is a functional unit of DNA consisting of two or more basic parts assembled together. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_I13507">BBa_I13507</a> is an example of a composite part, consisting of an RBS, a protein coding region for a red fluorescent protein, and a terminator.
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                    </p>
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</p>
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                <div class="standard-page-content-section-last">
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<p>New composite BioBrick devices can be made by combining existing BioBrick Parts (like Inverters, Amplifiers, Smell Generators, Protein Balloon Generators, Senders, Receivers, Actuators, and so on).</p>
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                    <div class="standard-page-content-title"><div id="reporterconstructs">Reporter Constructs</div></div>
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</div>
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                    <hr class="yellow-accent-line-left">
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                    <div class="standard-page-content-subheading"></div>
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<div class="highlight decoration_background">
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                        These two composite parts were used to demonstrate the oscillatory nature of our genetic circuit. While incubated at 37 degrees Celsius, cells expressing the low pass reporter construct would express sfGFP (the reporter molecule), while cells expressing the high pass reporter construct would cease expression of sfGFP (normally constitutively expressed)
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<h3>Note</h3>
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                    </p>
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<p>This page should list all the composite parts your team has 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. Remember judges will only look at the first part in the list for the Best Composite Part award, so put your best part first!</p>
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                    <div class="standard-page-content-subheading">BBa_K2561005: High Pass Reporter Construct</div>
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</div>
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                    <br>
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</div>
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                    <p class="standard-page-content-text">A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream reporter products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of a sfGFP reporter molecule that is normally constitutively expressed under the pTet promoter (BBa_R0040). TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations. </p>
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/f/fc/T--Cornell--demonstrateFigure1b.png" id="composite-img">
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                <div class="standard-page-content-section">
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                    <div class="standard-page-content-subheading">BBa_K2561003: Low Pass Reporter Construct</div>
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<div class="column full_size">
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                    <br>
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<h3>Best Composite Part Special Prize</h3>
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                    <p class="standard-page-content-text">A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (Sigma F, BBa_K2561001) that in turn directs the expression of downstream reporter products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The Sigma F produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence (to direct expression of sfGFP, a reporter molecule. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations.</p>
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/b/bd/T--Cornell--demonstrateFigure1a.png" id="composite-img">
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<p>To be eligible for this award, this part 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. If you have a part you wish to nominate your team for this <a href="https://2018.igem.org/Judging/Awards">special prize</a>, make sure you add your part number to your <a href="https://2018.igem.org/Judging/Judging_Form">judging form</a> and delete the box at the top of this page.
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                </div>
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                <div class="standard-page-content-section-last">
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<br><br>
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                    <div class="standard-page-content-title"><div id="combinationconstructs">Combination Constructs</div></div>
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<b>Please note:</b> Judges will only look at the first part number you list, so please only enter ONE (1) part number in the judging form for this prize. </p>
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                    <hr class="yellow-accent-line-left">
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/5/57/T--Cornell--CombinationConstructSchematics.jpg" id="composite-img" style="height: 250px;">
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                    <p class="standard-page-content-text">
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                        The following two parts were designed to be be cotransformed into the same cell to control expression of a gene of interest under the control of the sigma-54-dependent <i>hrpL</i> promoter.
 +
                    </p>
 +
                    <div class="standard-page-content-subheading">BBa_K2561006: High Pass Combination Construct (AND gate)</div>
 +
                    <p class="standard-page-content-text">
 +
                        A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream gene products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of HrpR, an enhancer-binding protein that forms a heteromeric complex with HrpS to regulate expression of the sigma-54-dependent <i>hrpL</i> promoter. TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations. This part should be used in conjunction with the Low Pass Combination Construct (AND gate) part (BBa_K2561004).
 +
                    </p>
 +
                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/5/51/T--Cornell--demonstrateFigure1d.png" id="composite-img" style="padding-bottom: 0">
 +
                    <div class="standard-page-content-subheading">BBa_K2561004: Low Pass Combination Construct (AND gate)</div>
 +
                    <p class="standard-page-content-text">
 +
                        A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (SigmaF) that in turn directs the expression of downstream gene products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The SigmaF produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence to direct expression of HrpS, an enhancer-binding protein that forms a heteromeric complex with HrpR to regulate expression of the sigma-54-dependent <i>hrpL</i> promoter. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations.This part should be used in conjunction with the High Pass Combination Construct (AND gate) part (BBa_K2561006).
 +
                    </p>
 +
                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/a/ab/T--Cornell--demonstrateFigure1c.png" id="composite-img" style="padding-bottom: 0">
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Latest revision as of 02:08, 18 October 2018

Team:Cornell/Composite - 2018.igem.org

Composite Part

This year, we are proud to present a variety of parts that ultimately allow for control of gene expression using specific frequency-based signals. The final gene products of these constructs can be replaced by any gene of interest, making these parts a versatile platform for precisely regulating a gene’s expression in multi-plasmid or construct systems. We have submitted BBa_K2561003 for consideration for best composite part.

Reporter Constructs

These two composite parts were used to demonstrate the oscillatory nature of our genetic circuit. While incubated at 37 degrees Celsius, cells expressing the low pass reporter construct would express sfGFP (the reporter molecule), while cells expressing the high pass reporter construct would cease expression of sfGFP (normally constitutively expressed)

BBa_K2561005: High Pass Reporter Construct

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream reporter products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of a sfGFP reporter molecule that is normally constitutively expressed under the pTet promoter (BBa_R0040). TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations.

BBa_K2561003: Low Pass Reporter Construct

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (Sigma F, BBa_K2561001) that in turn directs the expression of downstream reporter products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The Sigma F produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence (to direct expression of sfGFP, a reporter molecule. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations.

Combination Constructs

The following two parts were designed to be be cotransformed into the same cell to control expression of a gene of interest under the control of the sigma-54-dependent hrpL promoter.

BBa_K2561006: High Pass Combination Construct (AND gate)

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream gene products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of HrpR, an enhancer-binding protein that forms a heteromeric complex with HrpS to regulate expression of the sigma-54-dependent hrpL promoter. TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations. This part should be used in conjunction with the Low Pass Combination Construct (AND gate) part (BBa_K2561004).

BBa_K2561004: Low Pass Combination Construct (AND gate)

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (SigmaF) that in turn directs the expression of downstream gene products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The SigmaF produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence to direct expression of HrpS, an enhancer-binding protein that forms a heteromeric complex with HrpR to regulate expression of the sigma-54-dependent hrpL promoter. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations.This part should be used in conjunction with the High Pass Combination Construct (AND gate) part (BBa_K2561006).