Difference between revisions of "Team:Fudan-TSI/Part Collection"
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| − | <i class="fa fa-navicon" style="font-size: 24px"></i> | + | <i class="fa fa-navicon" style="font-size:24px"></i> |
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| − | <p style="width: 100%;text-align: center | + | <p class="flow-text" style="width:100%;text-align:center"><span class="white-text">Applied Design</span></p> |
</div></li> | </div></li> | ||
<li> | <li> | ||
<ul class="collapsible expandable"> | <ul class="collapsible expandable"> | ||
| − | <li>On this page</li> | + | <li class="onThisPageNav"><span>On this page</span></li> |
<li class="onThisPageNav"><a href="#section1">div with id section1</a></li> | <li class="onThisPageNav"><a href="#section1">div with id section1</a></li> | ||
<li class="onThisPageNav"><a href="#section2">div with id section2</a></li> | <li class="onThisPageNav"><a href="#section2">div with id section2</a></li> | ||
<li class="onThisPageNav"><a href="#section3">div with id section1</a></li> | <li class="onThisPageNav"><a href="#section3">div with id section1</a></li> | ||
| − | <li>Team: Fudan-TSI</li> | + | <li class="onThisPageNav"><span>Team: Fudan-TSI</span></li> |
| − | <li><div class="collapsible-header">Project</div> | + | <li><div class="collapsible-header"><span>Project</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Description">Background</a></li> | <li><a href="/Team:Fudan-TSI/Description">Background</a></li> | ||
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</ul></div> | </ul></div> | ||
</li> | </li> | ||
| − | <li><div class="collapsible-header">Results</div> | + | <li><div class="collapsible-header"><span>Results</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Results#ReverseTranscription">Reverse Transcription</a></li> | <li><a href="/Team:Fudan-TSI/Results#ReverseTranscription">Reverse Transcription</a></li> | ||
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</ul></div> | </ul></div> | ||
</li> | </li> | ||
| − | <li><div class="collapsible-header">Model</div> | + | <li><div class="collapsible-header"><span>Model</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Model">Modeling</a></li> | <li><a href="/Team:Fudan-TSI/Model">Modeling</a></li> | ||
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</ul></div> | </ul></div> | ||
</li> | </li> | ||
| − | <li><div class="collapsible-header">Parts</div> | + | <li><div class="collapsible-header"><span>Parts</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Basic_Part">Basic parts</a></li> | <li><a href="/Team:Fudan-TSI/Basic_Part">Basic parts</a></li> | ||
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</ul></div> | </ul></div> | ||
</li> | </li> | ||
| − | <li><div class="collapsible-header">Outreach</div> | + | <li><div class="collapsible-header"><span>Outreach</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Public_Engagement">Education & Public engagement</a></li> | <li><a href="/Team:Fudan-TSI/Public_Engagement">Education & Public engagement</a></li> | ||
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</ul></div> | </ul></div> | ||
</li> | </li> | ||
| − | <li><div class="collapsible-header">Team</div> | + | <li><div class="collapsible-header"><span>Team</span></div> |
<div class="collapsible-body"><ul> | <div class="collapsible-body"><ul> | ||
<li><a href="/Team:Fudan-TSI/Team">Members</a></li> | <li><a href="/Team:Fudan-TSI/Team">Members</a></li> | ||
<li><a href="/Team:Fudan-TSI/Attributions">Attributions</a></li> | <li><a href="/Team:Fudan-TSI/Attributions">Attributions</a></li> | ||
<li><a href="https://2018.igem.org/Team:Fudan/Heritage" target=_blank>Heritage</a></li> | <li><a href="https://2018.igem.org/Team:Fudan/Heritage" target=_blank>Heritage</a></li> | ||
| − | |||
</ul></div> | </ul></div> | ||
</li> | </li> | ||
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<div id="figureBannerTitle" class="hide-on-small-only"> | <div id="figureBannerTitle" class="hide-on-small-only"> | ||
<h1>Part collection</h1> | <h1>Part collection</h1> | ||
| − | <p><span>Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells.</span></p> | + | <p class="flow-text"><span>Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells.</span></p> |
</div> | </div> | ||
<div class="hide-on-small-only"> | <div class="hide-on-small-only"> | ||
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<main> | <main> | ||
<div class="section container"> | <div class="section container"> | ||
| − | <p>Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells. These parts were made as BioBricks, and are NOT ready for transfecting mammalian cells. Plasmids with these parts in eukaryotic expression backbone are available upon request. | + | <p class="flow-text">Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells. These parts were made as BioBricks, and are NOT ready for transfecting mammalian cells. Plasmids with these parts in eukaryotic expression backbone are available upon request. |
</p> | </p> | ||
| − | <p>Our parts collection contains <b>all essential components</b> of our ENABLE toolbox, that could also be divided into several sets, enabling others to readily utilize them to improve their existing genetic circuits. | + | <p class="flow-text">Our parts collection contains <b>all essential components</b> of our ENABLE toolbox, that could also be divided into several sets, enabling others to readily utilize them to improve their existing genetic circuits. |
</p> | </p> | ||
| − | <p>Following our 3-layer design principle, all ENABLE components have been well characterized, including our SynNotch receptors, Amplifiers and Combiners. In the collection, we have provided 7 versions of SynNotch receptors with (1) different combinations of extracellular domains which can bind to different antigens with different affinities, (2) transmembrane domains with varying cleavage efficiency and (3) intracellular domains executing diverse transcriptional amplifications. | + | <p class="flow-text">Following our 3-layer design principle, all ENABLE components have been well characterized, including our SynNotch receptors, Amplifiers and Combiners. In the collection, we have provided 7 versions of SynNotch receptors with (1) different combinations of extracellular domains which can bind to different antigens with different affinities, (2) transmembrane domains with varying cleavage efficiency and (3) intracellular domains executing diverse transcriptional amplifications. |
</p> | </p> | ||
| − | <p>We have provided a set of transcriptional amplifiers including zinc finger-based, TEV protease-based and split intein-based ones, allowing others to <b>build their own genetic circuits</b>, preferably through transcription (we have tested), but not limited to (we have not tested). Besides, a set of combiners with different copies of response elements are provided, which makes it <b>easy for others to tune the activation or repression threshold</b>. This feature was experimental confirmed previously and mathematically <a href="/Team:Fudan-TSI/Model#Transcriptional_Amplifier" target="_blank">modeled</a> in our 3-layer design again this year. Our modeling process could be used by others to predict their own genetic circuits before any wet-lab experiments. | + | <p class="flow-text">We have provided a set of transcriptional amplifiers including zinc finger-based, TEV protease-based and split intein-based ones, allowing others to <b>build their own genetic circuits</b>, preferably through transcription (we have tested), but not limited to (we have not tested). Besides, a set of combiners with different copies of response elements are provided, which makes it <b>easy for others to tune the activation or repression threshold</b>. This feature was experimental confirmed previously and mathematically <a href="/Team:Fudan-TSI/Model#Transcriptional_Amplifier" target="_blank">modeled</a> in our 3-layer design again this year. Our modeling process could be used by others to predict their own genetic circuits before any wet-lab experiments. |
</p> | </p> | ||
| − | <p> | + | <p class="flow-text"> |
Each part (sometimes two combined) constitutes one of our ENABLE module, which has its unique function. Yet they consolidate together to create all the 16 transmembrane binary logic gates for mammalian cells. | Each part (sometimes two combined) constitutes one of our ENABLE module, which has its unique function. Yet they consolidate together to create all the 16 transmembrane binary logic gates for mammalian cells. | ||
</p> | </p> | ||
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<div id="section1" class="section container scrolSpy"> | <div id="section1" class="section container scrolSpy"> | ||
<h2>SynNotch receptors set</h2> | <h2>SynNotch receptors set</h2> | ||
| − | <p>We have provided 7 versions of SynNotch receptors. Each of them can transduce signal from out of the cell via a contact-dependent antigen-stimulated cleavage process. When expressing two SynNotch receptors with two different extracellular domains recognizing two types of antigens, the cell is able to accept dual inputs. The chosen nanobodies are highly specific against their antigens, and the chosen intracellular domains are transcriptionally orthogonal. For more specific details, please refer to our parts listed below.</p> | + | <p class="flow-text">We have provided 7 versions of SynNotch receptors. Each of them can transduce signal from out of the cell via a contact-dependent antigen-stimulated cleavage process. When expressing two SynNotch receptors with two different extracellular domains recognizing two types of antigens, the cell is able to accept dual inputs. The chosen nanobodies are highly specific against their antigens, and the chosen intracellular domains are transcriptionally orthogonal. For more specific details, please refer to our parts listed below.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
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<div id="section2" class="section container scrolSpy"> | <div id="section2" class="section container scrolSpy"> | ||
<h2>Transcriptional amplifiers set</h2> | <h2>Transcriptional amplifiers set</h2> | ||
| − | <p>Last year, we have constructed <a href="https://2017.igem.org/Team:Fudan/Part_Collection" target="_blank">3 zinc finger-based transcription repressors</a>. This year, we have expanded our zinc finger-based transcription factors library by adding 3 more zinc finger-based transcription activators.</p> | + | <p class="flow-text">Last year, we have constructed <a href="https://2017.igem.org/Team:Fudan/Part_Collection" target="_blank">3 zinc finger-based transcription repressors</a>. This year, we have expanded our zinc finger-based transcription factors library by adding 3 more zinc finger-based transcription activators.</p> |
| − | <p> | + | <p class="flow-text"> |
Although we prefer to use eight copies of response elements (RE) for a balance between not-too-hard molecular cloning and sufficient signal-to-noise ratio, we have provided 4 promotors with different repeats of response elements, <b>allowing others to explore and tune their own transcriptional amplifiers</b>. <a href="/Team:Fudan-TSI/Model#Transcriptional_Amplifer" target="_blank">Latest modeling</a> strongly supports our experimental preference, showing that eight copies of response elements can render the best result in line with our needs.</p> | Although we prefer to use eight copies of response elements (RE) for a balance between not-too-hard molecular cloning and sufficient signal-to-noise ratio, we have provided 4 promotors with different repeats of response elements, <b>allowing others to explore and tune their own transcriptional amplifiers</b>. <a href="/Team:Fudan-TSI/Model#Transcriptional_Amplifer" target="_blank">Latest modeling</a> strongly supports our experimental preference, showing that eight copies of response elements can render the best result in line with our needs.</p> | ||
<div class="tableHolder"> | <div class="tableHolder"> | ||
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</table> | </table> | ||
</div> | </div> | ||
| − | <p>As is stated above, we have provided three types of transcriptional-based Amplifiers, including zinc finger-based, TEV protease-based and split intein-based. These Amplifiers must be used with our Combiners to execute designed binary logic function.</p> | + | <p class="flow-text">As is stated above, we have provided three types of transcriptional-based Amplifiers, including zinc finger-based, TEV protease-based and split intein-based. These Amplifiers must be used with our Combiners to execute designed binary logic function.</p> |
</div> | </div> | ||
<div id="section3" class="section container scrolSpy"> | <div id="section3" class="section container scrolSpy"> | ||
<h2>Amplifiers and Combiners work together to execute binary computation</h2> | <h2>Amplifiers and Combiners work together to execute binary computation</h2> | ||
<h3>Zinc finger-based</h3> | <h3>Zinc finger-based</h3> | ||
| − | <p>For simple binary logic function, such as A gate, NOT A gate, OR gate, NOR gate, and NIMPLY gate, placing response elements upstream and downstream of the promoter is sufficient. For more details, please visit our <a href="/Team:Fudan-TSI/Results" target=”_blank”>results page</a>. | + | <p class="flow-text">For simple binary logic function, such as A gate, NOT A gate, OR gate, NOR gate, and NIMPLY gate, placing response elements upstream and downstream of the promoter is sufficient. For more details, please visit our <a href="/Team:Fudan-TSI/Results" target=”_blank”>results page</a>. |
</p> | </p> | ||
<div class="tableHolder"> | <div class="tableHolder"> | ||
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</div> | </div> | ||
<h3> TEV protease-based</h3> | <h3> TEV protease-based</h3> | ||
| − | <p>For IMPLY gate, we placed TEV protease-controlled destroyable nuclear localization sequence between zinc finger DNA binding domain (DBD) and transcription factor (TF). In the presence of intracellular TEV protease, DBD-TF would be cleaved, thus destroying its transcriptional regulation function and efficiently blocking its signal. For more details, please visit our <a href="/Team:Fudan-TSI/Results" target=”_blank”>results page</a>.</p> | + | <p class="flow-text">For IMPLY gate, we placed TEV protease-controlled destroyable nuclear localization sequence between zinc finger DNA binding domain (DBD) and transcription factor (TF). In the presence of intracellular TEV protease, DBD-TF would be cleaved, thus destroying its transcriptional regulation function and efficiently blocking its signal. For more details, please visit our <a href="/Team:Fudan-TSI/Results" target=”_blank”>results page</a>.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
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</div> | </div> | ||
<h3>Split intein-based</h3> | <h3>Split intein-based</h3> | ||
| − | <p>We built the more complex binary logic gates, such as XOR and XNOR gate, using split intein-based Amplifiers. Utilizing intein's ability to fuse the split ends of a transcription factor together, we're able to present these logic gates consistent with our 3-layer design principle. For more details, please visit our <a href="/Team:Fudan-TSI/Results" target=”_blank”>results page</a>.</p> | + | <p class="flow-text">We built the more complex binary logic gates, such as XOR and XNOR gate, using split intein-based Amplifiers. Utilizing intein's ability to fuse the split ends of a transcription factor together, we're able to present these logic gates consistent with our 3-layer design principle. For more details, please visit our <a href="/Team:Fudan-TSI/Results" target=”_blank”>results page</a>.</p> |
<div class="tableHolder"> | <div class="tableHolder"> | ||
<table class="striped"> | <table class="striped"> | ||
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</table> | </table> | ||
</div> | </div> | ||
| − | <p>Here is our 3-layer design of transmembrane binary logic gates. On the left lying a truth table which indicates the inputs and outputs of the according logic computation. What is set on the right is how it works. The double crossline on the top represents the plasma membrane. A and B signify the signal input, which are surface-expressed EGFP and surface-expressed CD19 in our system. What are embed on the membrane are our SynNotch receptors, the intracellular domain of which are transcriptional activators, which are tTAA and GV2 in our assay. The large rectangle represents the nucleus. The elongated rectangle with an array before represents the genetic circuit that are under control of the transcriptional factors. Squares in groups of three represent transcriptional factors that are amplified by the amplifier layer. You can swipe the bar to detect other gates by yourself.</p> | + | <p class="flow-text">Here is our 3-layer design of transmembrane binary logic gates. On the left lying a truth table which indicates the inputs and outputs of the according logic computation. What is set on the right is how it works. The double crossline on the top represents the plasma membrane. A and B signify the signal input, which are surface-expressed EGFP and surface-expressed CD19 in our system. What are embed on the membrane are our SynNotch receptors, the intracellular domain of which are transcriptional activators, which are tTAA and GV2 in our assay. The large rectangle represents the nucleus. The elongated rectangle with an array before represents the genetic circuit that are under control of the transcriptional factors. Squares in groups of three represent transcriptional factors that are amplified by the amplifier layer. You can swipe the bar to detect other gates by yourself.</p> |
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<a href="#!"><img alt="project summary" src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a> | <a href="#!"><img alt="project summary" src="https://static.igem.org/mediawiki/2018/9/96/T--Fudan--X.svg"></a> | ||
<div class="container"> | <div class="container"> | ||
| − | < | + | <h4 style="margin:0;padding:10px 0;">Project Summary</h4> |
| − | <p style="margin: 0">Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for <i>in vivo</i> continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the <i>in vivo</i> evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology. | + | <p class="flow-text" style="margin:0">Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for <i>in vivo</i> continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the <i>in vivo</i> evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology. |
</p> | </p> | ||
</div> | </div> | ||
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<a href="#!" id="abstractBtn" class="btn"> | <a href="#!" id="abstractBtn" class="btn"> | ||
| − | <i class="fa fa-sticky-note" style="font-size: 30px;line-height: 50px"></i> | + | <i class="fa fa-sticky-note" style="font-size:30px;line-height:50px"></i> |
</a> | </a> | ||
<a href="#FudanWrapper" class="btn"> | <a href="#FudanWrapper" class="btn"> | ||
| − | <i class="fa fa-angle-up" style="font-size: 48px;line-height: 45px"></i> | + | <i class="fa fa-angle-up" style="font-size:48px;line-height:45px"></i> |
</a> | </a> | ||
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</a><a href="http://www.yfc.cn/en/" target="_blank"><img class="col s3 m6 l3" style="padding: 0.15rem 0.9rem;" alt="Yunfeng Capital" src="https://static.igem.org/mediawiki/2018/e/e2/T--Fudan--yunfengLogo.png"> | </a><a href="http://www.yfc.cn/en/" target="_blank"><img class="col s3 m6 l3" style="padding: 0.15rem 0.9rem;" alt="Yunfeng Capital" src="https://static.igem.org/mediawiki/2018/e/e2/T--Fudan--yunfengLogo.png"> | ||
</a> | </a> | ||
| − | <h3 class="col s12" style="text-align: left; color: rgba(255, 255, 255, 0.8); font-size: | + | <h3 class="col s12" style="text-align: left; color: rgba(255, 255, 255, 0.8); font-size:12.5px">R-Evolution: an <i>in vivo</i> sequence-specific toolbox for continuous mutagenesis</h3> |
</div> | </div> | ||
<div id="usefulLinks" class="col m9 s12 row"> | <div id="usefulLinks" class="col m9 s12 row"> | ||
Revision as of 07:11, 17 September 2019
<script src="https://code.jquery.com/jquery-1.11.3.min.js"></script>
Applied Design
Our parts collection provides an extensive toolbox for researchers to construct their own transmembrane logic gates in mammalian cells. These parts were made as BioBricks, and are NOT ready for transfecting mammalian cells. Plasmids with these parts in eukaryotic expression backbone are available upon request.
Our parts collection contains all essential components of our ENABLE toolbox, that could also be divided into several sets, enabling others to readily utilize them to improve their existing genetic circuits.
Following our 3-layer design principle, all ENABLE components have been well characterized, including our SynNotch receptors, Amplifiers and Combiners. In the collection, we have provided 7 versions of SynNotch receptors with (1) different combinations of extracellular domains which can bind to different antigens with different affinities, (2) transmembrane domains with varying cleavage efficiency and (3) intracellular domains executing diverse transcriptional amplifications.
We have provided a set of transcriptional amplifiers including zinc finger-based, TEV protease-based and split intein-based ones, allowing others to build their own genetic circuits, preferably through transcription (we have tested), but not limited to (we have not tested). Besides, a set of combiners with different copies of response elements are provided, which makes it easy for others to tune the activation or repression threshold. This feature was experimental confirmed previously and mathematically modeled in our 3-layer design again this year. Our modeling process could be used by others to predict their own genetic circuits before any wet-lab experiments.
Each part (sometimes two combined) constitutes one of our ENABLE module, which has its unique function. Yet they consolidate together to create all the 16 transmembrane binary logic gates for mammalian cells.
SynNotch receptors set
We have provided 7 versions of SynNotch receptors. Each of them can transduce signal from out of the cell via a contact-dependent antigen-stimulated cleavage process. When expressing two SynNotch receptors with two different extracellular domains recognizing two types of antigens, the cell is able to accept dual inputs. The chosen nanobodies are highly specific against their antigens, and the chosen intracellular domains are transcriptionally orthogonal. For more specific details, please refer to our parts listed below.
| BioBrick ID | Protein Name | Recognized Antigen (affinity) | Transmembrane Domain | Intracellular Domain | Sequence Verification |
|---|---|---|---|---|---|
| BBa_K2549016 | LaG17-mN1c-tTAA | GFP (low) | mouse Notch1 core | tTA advance | 016.ab1 |
| BBa_K2549017 | LaG17-mN1ce-tTAA | GFP (low) | mouse Notch1 extended core | tTA advance | 017.ab1 |
| BBa_K2549018 | LaG16-mN1ce-tTAA | GFP (high) | mouse Notch1 extended core | tTA advance | 018.ab1 |
| BBa_K2549019 | LaG16-2-mN1c-tTAA | GFP (ultrahigh) | mouse Notch1 core | tTA advance | 019.ab1 |
| BBa_K2549020 | LaG16-2-mN1c-GV2 | GFP (ultrahigh) | mouse Notch1 core | Gal4-VP64 | 020.ab1 |
| BBa_K2549021 | αCD19-mN1c-tTAA | CD19 | mouse Notch1 core | Gal4-VP64 | 021.ab1 |
| BBa_K2549022 | αCD19-mN1c-GV2 | CD19 | mouse Notch1 core | Gal4-VP64 | 022.ab1 |
Transcriptional amplifiers set
Last year, we have constructed 3 zinc finger-based transcription repressors. This year, we have expanded our zinc finger-based transcription factors library by adding 3 more zinc finger-based transcription activators.
Although we prefer to use eight copies of response elements (RE) for a balance between not-too-hard molecular cloning and sufficient signal-to-noise ratio, we have provided 4 promotors with different repeats of response elements, allowing others to explore and tune their own transcriptional amplifiers. Latest modeling strongly supports our experimental preference, showing that eight copies of response elements can render the best result in line with our needs.
| BioBrick ID | Protein Name | DNA Binding Domain | Transcriptional | Sequence Verification |
|---|---|---|---|---|
| BBa_K2549023 | ZF21.16-VP64 | ZF21.16 | activation | 023.ab1 |
| BBa_K2549024 | ZF42.10-VP64 | ZF42.10 | activation | 024.ab1 |
| BBa_K2549025 | ZF43.8-VP64 | ZF43.8 | activation | 025.ab1 |
| BBa_K2446039 | ZF21.16-KRAB | ZF21.16 | repression | 039.abl |
| BBa_K2446040 | ZF42.10-KRAB | ZF42.10 | repression | 040.abl |
| BBa_K2446041 | ZF43.8-KRAB | ZF43.8 | repression | 041.abl |
| BioBrick ID | DNA Name | Usage | Sequence Verification |
|---|---|---|---|
| BBa_K2549032 | 4*ZF21.16-minCMV-2*ZF43.8 | 4 copies of RE for ZF21.16 transcriptional activator to bind, and 2 copies of RE for ZF43.8 transcriptional repressor to bind | 032.ab1 |
| BBa_K2549033 | 6*ZF21.16-minCMV-2*ZF43.8 | 6 copies of RE for ZF21.16 transcriptional activator to bind, and 2 copies of RE for ZF43.8 transcriptional repressor to bind | 033.ab1 |
| BBa_K2549034 | 8*ZF21.16-minCMV-2*ZF43.8 | 8 copies of RE for ZF21.16 transcriptional activator to bind, and 2 copies of RE for ZF43.8 transcriptional repressor to bind | 034.ab1 |
| BBa_K2549035 | 8*ZF21.16-minCMV-8*ZF43.8 | 8 copies of RE for ZF21.16 transcriptional activator to bind, and 8 copies of RE for ZF43.8 transcriptional repressor to bind | 035.ab1 |
As is stated above, we have provided three types of transcriptional-based Amplifiers, including zinc finger-based, TEV protease-based and split intein-based. These Amplifiers must be used with our Combiners to execute designed binary logic function.
Amplifiers and Combiners work together to execute binary computation
Zinc finger-based
For simple binary logic function, such as A gate, NOT A gate, OR gate, NOR gate, and NIMPLY gate, placing response elements upstream and downstream of the promoter is sufficient. For more details, please visit our results page.
| BioBrick ID | Combiner Name | Usage | Sequence Verification |
|---|---|---|---|
| BBa_K2549026 | 8*ZF21.16-minCMV | 8 copies of RE for ZF21.16 transcriptional activator to bind | 026.ab1 |
| BBa_K2549027 | 8*ZF42.10-minCMV | 8 copies of RE for ZF42.10 transcriptional activator to bind | 027.ab1 |
| BBa_K2549028 | 8*ZF43.8-minCMV | 8 copies of RE for ZF43.8 transcriptional activator to bind | 028.ab1 |
| BBa_K2549029 | 8*ZF21.16-CMV | 8 copies of RE for ZF21.16 transcriptional activator to bind | 029.ab1 |
| BBa_K2549030 | 8*ZF42.10-CMV | 8 copies of RE for ZF42.10 transcriptional activator to bind | 030.ab1 |
| BBa_K2549031 | 8*ZF43.8-CMV | 8 copies of RE for ZF43.8 transcriptional activator to bind | 031.ab1 |
TEV protease-based
For IMPLY gate, we placed TEV protease-controlled destroyable nuclear localization sequence between zinc finger DNA binding domain (DBD) and transcription factor (TF). In the presence of intracellular TEV protease, DBD-TF would be cleaved, thus destroying its transcriptional regulation function and efficiently blocking its signal. For more details, please visit our results page.
| BioBrick ID | Protein Name | Sequence Verification |
|---|---|---|
| BBa_K2549039 | VP64-dNLS-ZF21.16 | 039.ab1 |
| BBa_K2549040 | KRAB-dNLS-ZF21.16 | 040.ab1 |
| BBa_K2549041 | NLS-TEVp | 041.ab1 |
Split intein-based
We built the more complex binary logic gates, such as XOR and XNOR gate, using split intein-based Amplifiers. Utilizing intein's ability to fuse the split ends of a transcription factor together, we're able to present these logic gates consistent with our 3-layer design principle. For more details, please visit our results page.
| BioBrick ID | Protein Name | Sequence Verification |
|---|---|---|
| BBa_K2549036 | VP64-ZF21.16N-CfaN | 036.ab1 |
| BBa_K2549037 | KRAB-ZF21.16N-CfaN | 037.ab1 |
| BBa_K2549038 | CfaC-ZF21.16C-NLS | 038.ab1 |
| BBa_K2549042 | NLS-TEVpN-CfaN | 042.ab1 |
| BBa_K2549043 | CfaC-TEVpC | 043.ab1 |
ENABLE - 16 logic gates for transmembrane signaling
| Gate | Intracellular Domain of the Receptor | the Amplifier | the Combiner |
|---|---|---|---|
| TRUE | tTAA + Gal4-VP64 | / | CMV-d2EGFP |
| FALSE | tTAA + Gal4-VP64 | / | / |
| A | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-VP64 | 8*ZF21.16-minCMV-d2EGFP |
| B | tTAA + Gal4-VP64 | 4*UAS-ZF21.16-VP64 | 8*ZF21.16-minCMV-d2EGFP |
| NOT A | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-KRAB | 8*ZF21.16-CMV-d2EGFP |
| NOT B | tTAA + Gal4-VP64 | 4*UAS-ZF21.16-KRAB | 8*ZF21.16-CMV-d2EGFP |
| OR | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-VP64 4*UAS-minCMV-ZF21.16-VP64 | 8*ZF21.16-minCMV-d2EGFP |
| NOR | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-KRAB 4*UAS-minCMV-ZF21.16-KRAB | 8*ZF21.16-CMV-d2EGFP |
| XOR | tTAA + Gal4-VP64 | TRE3GV-VP64-dNLS-ZF21.16-T2A-NLS-TEVpN-CfaN 4*UAS-minCMV-CfaC-TEVpC-T2A-VP64-dNLS-ZF21.16 | 8*ZF21.16-minCMV-d2EGFP |
| AND | tTAA + Gal4-VP64 | TRE3GV-VP64-ZF21.16N-CfaN 4*UAS-minCMV-CfaC-ZF21.16C-NLS | 8*ZF21.16-minCMV-d2EGFP |
| NAND | tTAA + Gal4-VP64 | TRE3GV-KRAB-ZF21.16N-CfaN 4*UAS-minCMV-CfaC-ZF21.16C-NLS | 8*ZF21.16-CMV-d2EGFP |
| A IMPLY B | tTAA + Gal4-VP64 | TRE3GV-KRAB-dNLS-ZF21.16 4*UAS-minCMV-NLS-TEVp | 8*ZF21.16-CMV-d2EGFP |
| B IMPLY A | tTAA + Gal4-VP64 | 4*UAS-minCMV-KRAB-dNLS-ZF21.16 TRE3GV-NLS-TEVp | 8*ZF21.16-CMV-d2EGFP |
| A NIMPLY B | tTAA + Gal4-VP64 | TRE3GV-ZF21.16-VP64 4*UAS-minCMV-ZF43.8-KRAB | 8*ZF21.16-minCMV-2*ZF43.8-d2EGFP |
| B NIMPLY A | tTAA + Gal4-VP64 | 4*UAS-minCMV-ZF21.16-VP64 TRE3GV-ZF43.8-KRAB | 8*ZF21.16-minCMV-2*ZF43.8-d2EGFP |
| XNOR | tTAA + Gal4-VP64 | TRE3GV-KRAB-dNLS-ZF21.16-T2A-NLS-TEVpN-CfaN 4*UAS-minCMV-CfaC-TEVpC-T2A-KRAB-dNLS-ZF21.16 | 8*ZF21.16-CMV-d2EGFP |
Here is our 3-layer design of transmembrane binary logic gates. On the left lying a truth table which indicates the inputs and outputs of the according logic computation. What is set on the right is how it works. The double crossline on the top represents the plasma membrane. A and B signify the signal input, which are surface-expressed EGFP and surface-expressed CD19 in our system. What are embed on the membrane are our SynNotch receptors, the intracellular domain of which are transcriptional activators, which are tTAA and GV2 in our assay. The large rectangle represents the nucleus. The elongated rectangle with an array before represents the genetic circuit that are under control of the transcriptional factors. Squares in groups of three represent transcriptional factors that are amplified by the amplifier layer. You can swipe the bar to detect other gates by yourself.
Project Summary
Mutation library generation is critical for biological and medical research, but current methods cannot mutate a specific sequence continuously without manual intervention. Here we present a toolbox for in vivo continuous mutation library construction. First, the target DNA is transcribed into RNA. Next, our reverse transcriptase reverts RNA into cDNA, during which the target is randomly mutated by enhanced error-prone reverse transcription. Finally, the mutated version replaces the original sequence through recombination. These steps will be carried out iteratively, generating a random mutation library of the target with high efficiency as mutations accumulate along with bacterial growth. Our toolbox is orthogonal and provides a wide range of applications among various species. R-Evolution could mutate coding sequences and regulatory sequences, which enables the in vivo evolution of individual proteins or multiple targets at a time, promotes high-throughput research, and serves as a foundational advance to synthetic biology.