Difference between revisions of "Team:Fudan/Parts Collection"
| Line 286: | Line 286: | ||
<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>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 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. | + | <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> | </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>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 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 <a href="" target="_blank">experimental confirmed previously(需要对应链接)</a> and mathematically <a href="https://2018.igem.org/Team:Fudan/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>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 <a href="" target="_blank">experimental confirmed previously(需要对应链接)</a> and mathematically <a href="https://2018.igem.org/Team:Fudan/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> | ||
| Line 372: | Line 372: | ||
<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>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> | ||
| − | 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. <a href="" target="_blank">Latest modeling(需要对应链接)</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="" target="_blank">Latest modeling(需要对应链接)</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"> | ||
<table class="striped"> | <table class="striped"> | ||
Revision as of 14:52, 17 October 2018
Parts collection
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(需要对应链接) 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(需要对应链接) 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(需要对应链接) 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 | SynNotch Intracellular Domain | Amplifier | 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-8*ZF43.8-d2EGFP |
| B NIMPLY A | tTAA + Gal4-VP64 | 4*UAS-minCMV-ZF21.16-VP64 TRE3GV-ZF43.8-KRAB |
8*ZF21.16-minCMV-8*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 |
Abstract
Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.