Difference between revisions of "Part:BBa K5237017"
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<body> | <body> | ||
<!-- Part summary --> | <!-- Part summary --> | ||
− | <section | + | <section> |
<h1>FRET-Acceptor: TetR-mScarlet-I</h1> | <h1>FRET-Acceptor: TetR-mScarlet-I</h1> | ||
<p>This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET | <p>This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET | ||
Line 40: | Line 47: | ||
This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements. | This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements. | ||
</p> | </p> | ||
− | <p> | + | <p> </p> |
</section> | </section> | ||
− | <div | + | <div class="toc" id="toc"> |
<div id="toctitle"> | <div id="toctitle"> | ||
<h1>Contents</h1> | <h1>Contents</h1> | ||
Line 54: | Line 61: | ||
<ul> | <ul> | ||
<li class="toclevel-2 tocsection-2.1"><a href="#2.1"><span class="tocnumber">2.1</span> <span | <li class="toclevel-2 tocsection-2.1"><a href="#2.1"><span class="tocnumber">2.1</span> <span | ||
− | class="toctext">TetR DNA | + | class="toctext">TetR DNA Binding Protein</span></a> |
</li> | </li> | ||
<li class="toclevel-2 tocsection-2.2"><a href="#2.2"><span class="tocnumber">2.2</span> <span | <li class="toclevel-2 tocsection-2.2"><a href="#2.2"><span class="tocnumber">2.2</span> <span | ||
Line 65: | Line 72: | ||
</li> | </li> | ||
<li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly | <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly | ||
− | and | + | and Part Evolution</span></a> |
</li> | </li> | ||
<li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span | <li class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span | ||
class="toctext">Results</span></a> | class="toctext">Results</span></a> | ||
</li> | </li> | ||
− | <li class="toclevel-1 tocsection- | + | <li class="toclevel-1 tocsection-6"><a href="#5"><span class="tocnumber">5</span> <span |
+ | class="toctext">Conclusion</span></a> | ||
+ | <li class="toclevel-1 tocsection-8"><a href="#6"><span class="tocnumber">6</span> <span | ||
class="toctext">References</span></a> | class="toctext">References</span></a> | ||
+ | </li> | ||
</li> | </li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
<section> | <section> | ||
+ | <p><br /><br /></p> | ||
<font size="5"><b>The PICasSO Toolbox </b> </font> | <font size="5"><b>The PICasSO Toolbox </b> </font> | ||
− | + | <div class="thumb" style="margin-top:10px;"></div> | |
− | <div class="thumb"></div> | + | <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" |
− | <div class="thumbinner" style="width:550px"><img alt="" | + | |
src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" | src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" | ||
− | style="width:99%;" | + | style="width:99%;" /> |
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
− | <i><b>Figure 1: | + | <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i> |
</div> | </div> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
<p> | <p> | ||
− | <br> | + | <br /> |
− | + | While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the <b>3D | |
− | + | spatial organization</b> of DNA is well-known to be an important layer of information encoding in | |
− | + | particular in eukaryotes, playing a crucial role in | |
− | + | gene regulation and hence | |
− | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a | + | cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the |
− | toolbox based on various DNA-binding proteins | + | genomic spatial |
− | + | architecture are limited, hampering the exploration of | |
+ | 3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a | ||
+ | <b>powerful | ||
+ | molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on | ||
+ | various DNA-binding proteins. | ||
</p> | </p> | ||
<p> | <p> | ||
The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | ||
− | re-programming | + | <b>re-programming |
− | + | of DNA-DNA interactions</b> using engineered "protein staples" in living cells. This enables | |
− | interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. | + | researchers to recreate naturally occurring alterations of 3D genomic |
− | Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and | + | interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for |
− | testing of new staples | + | artificial gene regulation and cell function control. |
− | + | Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic | |
+ | loci into | ||
+ | spatial proximity. | ||
+ | To unlock the system's full potential, we introduce versatile <b>chimeric CRISPR/Cas complexes</b>, | ||
+ | connected either at | ||
+ | the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are | ||
+ | referred to as protein- or Cas staples, respectively. Beyond its | ||
+ | versatility with regard to the staple constructs themselves, PICasSO includes <b>robust assay</b> systems to | ||
+ | support the engineering, optimization, and | ||
+ | testing of new staples <i>in vitro</i> and <i>in vivo</i>. Notably, the PICasSO toolbox was developed in a | ||
+ | design-build-test-learn <b>engineering cycle closely intertwining wet lab experiments and computational | ||
+ | modeling</b> and iterated several times, yielding a collection of well-functioning and -characterized | ||
+ | parts. | ||
</p> | </p> | ||
− | + | <p>At its heart, the PICasSO part collection consists of three categories. <br /><b>(i)</b> Our <b>DNA-binding | |
− | <p>At its heart, the PICasSO part collection consists of three categories. (i) Our <b>DNA-binding proteins</b> | + | proteins</b> |
include our | include our | ||
− | finalized | + | finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as |
− | new Cas staples in the future. We also include our | + | "half staples" that can be combined by scientists to compose entirely |
− | and can be further engineered to create alternative, simpler and more compact staples. (ii) As <b>functional | + | new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple |
− | + | and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for | |
− | consist of | + | successful stapling |
− | + | and can be further engineered to create alternative, simpler, and more compact staples. <br /> | |
− | + | <b>(ii)</b> As <b>functional elements</b>, we list additional parts that enhance and expand the | |
− | interkingdom conjugation system. | + | functionality of our Cas and |
− | + | Basic staples. These | |
− | + | consist of staples dependent on | |
− | + | cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific, | |
− | systems</b>. These include components of our established FRET-based proximity assay system, enabling users to | + | dynamic stapling <i>in vivo</i>. |
+ | We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into | ||
+ | target cells, including mammalian cells, | ||
+ | with our new | ||
+ | interkingdom conjugation system. <br /> | ||
+ | <b>(iii)</b> As the final category of our collection, we provide parts that underlie our <b>custom | ||
+ | readout | ||
+ | systems</b>. These include components of our established FRET-based proximity assay system, enabling | ||
+ | users to | ||
confirm | confirm | ||
− | accurate stapling. Additionally, we offer a complementary, application-oriented testing system | + | accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a |
− | + | luciferase reporter, which allows for straightforward experimental assessment of functional enhancer | |
+ | hijacking events | ||
+ | in mammalian cells. | ||
</p> | </p> | ||
<p> | <p> | ||
− | The following table gives a | + | The following table gives a comprehensive overview of all parts in our PICasSO toolbox. <mark |
− | + | style="background-color: #FFD700; color: black;">The highlighted parts showed | |
− | collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer their | + | exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> The other |
− | own custom Cas staples, enabling further optimization and innovation | + | parts in |
+ | the | ||
+ | collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer | ||
+ | their | ||
+ | own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome | ||
+ | engineering.<br /> | ||
</p> | </p> | ||
<p> | <p> | ||
− | <font size="4"><b>Our part collection includes:</b></font><br> | + | <font size="4"><b>Our part collection includes:</b></font><br /> |
</p> | </p> | ||
− | + | <table style="width: 90%; padding-right:10px;"> | |
− | <table style="width: 90%;"> | + | <td align="left" colspan="3"><b>DNA-Binding Proteins: </b> |
− | <td | + | Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i> |
− | + | </td> | |
− | + | ||
<tbody> | <tbody> | ||
<tr bgcolor="#FFD700"> | <tr bgcolor="#FFD700"> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | ||
− | <td> | + | <td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td> |
− | <td> | + | <td>Entry vector for simple fgRNA cloning via SapI</td> |
</tr> | </tr> | ||
− | <tr> | + | <tr bgcolor="#FFD700"> |
<td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td> | ||
− | <td>Staple | + | <td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td> |
− | <td>Staple subunit that can be combined to form a functional staple | + | <td>Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple |
+ | </td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr bgcolor="#FFD700"> |
<td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td> | ||
− | <td>Staple | + | <td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td> |
− | <td>Staple subunit that can be combined to form a functional staple | + | <td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | ||
− | <td>Cas | + | <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td> |
− | <td>Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands | + | <td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into |
+ | close | ||
+ | proximity | ||
</td> | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | ||
− | <td>Staple | + | <td>Staple Subunit: Oct1-DBD</td> |
− | <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br> | + | <td>Staple subunit that can be combined to form a functional staple, for example with TetR.<br /> |
Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td> | ||
− | <td>Staple | + | <td>Staple Subunit: TetR</td> |
− | <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br> | + | <td>Staple subunit that can be combined to form a functional staple, for example with Oct1.<br /> |
Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | Can also be combined with a fluorescent protein as part of the FRET proximity assay</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td> | ||
− | <td>Simple | + | <td>Simple Staple: TetR-Oct1</td> |
<td>Functional staple that can be used to bring two DNA strands in close proximity</td> | <td>Functional staple that can be used to bring two DNA strands in close proximity</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td> | ||
− | <td>Staple | + | <td>Staple Subunit: GCN4</td> |
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td> | ||
− | <td>Staple | + | <td>Staple Subunit: rGCN4</td> |
<td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td> | ||
− | <td>Mini | + | <td>Mini Staple: bGCN4</td> |
<td> | <td> | ||
Assembled staple with minimal size that can be further engineered</td> | Assembled staple with minimal size that can be further engineered</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
− | <td | + | <td align="left" colspan="3"><b>Functional Elements: </b> |
− | Protease cleavable peptide linkers and inteins are used to control and modify staples for further optimization | + | Protease-cleavable peptide linkers and inteins are used to control and modify staples for further |
− | for custom applications | + | optimization |
+ | for custom applications</td> | ||
<tbody> | <tbody> | ||
<tr bgcolor="#FFD700"> | <tr bgcolor="#FFD700"> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | ||
− | <td>Cathepsin B- | + | <td>Cathepsin B-cleavable Linker: GFLG</td> |
− | <td>Cathepsin B cleavable peptide linker | + | <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make |
+ | responsive | ||
staples</td> | staples</td> | ||
</tr> | </tr> | ||
Line 211: | Line 253: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | ||
<td>Cathepsin B Expression Cassette</td> | <td>Cathepsin B Expression Cassette</td> | ||
− | <td> | + | <td>Expression cassette for the overexpression of cathepsin B</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td> |
<td>Caged NpuN Intein</td> | <td>Caged NpuN Intein</td> | ||
− | <td> | + | <td>A caged NpuN split intein fragment that undergoes protein <i>trans</i>-splicing after protease |
− | + | activation, which can be used to create functionalized staple | |
+ | subunits</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td> |
<td>Caged NpuC Intein</td> | <td>Caged NpuC Intein</td> | ||
− | <td> | + | <td>A caged NpuC split intein fragment that undergoes protein <i>trans</i>-splicing after protease |
− | + | activation, which can be used to create functionalized staple | |
+ | subunits</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> |
− | <td> | + | <td>Fusion Guide RNA Processing Casette</td> |
− | <td>Processing | + | <td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for |
+ | multiplexed 3D | ||
+ | genome reprogramming</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td> |
<td>Intimin anti-EGFR Nanobody</td> | <td>Intimin anti-EGFR Nanobody</td> | ||
− | <td> | + | <td>Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for |
+ | large | ||
constructs</td> | constructs</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K4643003" target="_blank">BBa_K4643003</a></td> | ||
+ | <td>IncP Origin of Transfer</td> | ||
+ | <td>Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a | ||
+ | means of | ||
+ | delivery</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
− | <td | + | <td align="left" colspan="3"><b>Readout Systems: </b> |
− | FRET and enhancer recruitment to | + | FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and |
− | + | mammalian cells | |
+ | </td> | ||
<tbody> | <tbody> | ||
<tr bgcolor="#FFD700"> | <tr bgcolor="#FFD700"> | ||
− | <td><a href="https://parts.igem.org/Part: | + | <td><a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a></td> |
<td>FRET-Donor: mNeonGreen-Oct1</td> | <td>FRET-Donor: mNeonGreen-Oct1</td> | ||
− | <td> | + | <td>FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to |
+ | visualize | ||
+ | DNA-DNA | ||
proximity</td> | proximity</td> | ||
</tr> | </tr> | ||
Line 250: | Line 307: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td> | ||
<td>FRET-Acceptor: TetR-mScarlet-I</td> | <td>FRET-Acceptor: TetR-mScarlet-I</td> | ||
− | <td>Acceptor part for the FRET assay binding the TetR binding cassette | + | <td>Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize |
+ | DNA-DNA | ||
proximity</td> | proximity</td> | ||
</tr> | </tr> | ||
Line 256: | Line 314: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td> | ||
<td>Oct1 Binding Casette</td> | <td>Oct1 Binding Casette</td> | ||
− | <td>DNA sequence containing 12 Oct1 binding motifs, | + | <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET |
proximity assay</td> | proximity assay</td> | ||
</tr> | </tr> | ||
Line 262: | Line 320: | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | ||
<td>TetR Binding Cassette</td> | <td>TetR Binding Cassette</td> | ||
− | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET | + | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the |
+ | FRET | ||
proximity assay</td> | proximity assay</td> | ||
</tr> | </tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | ||
<td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td> | <td>Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64</td> | ||
− | <td>Readout system that responds to protease activity | + | <td>Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker |
− | </ | + | </td> |
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td> | ||
<td>NLS-Gal4-VP64</td> | <td>NLS-Gal4-VP64</td> | ||
− | <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking | + | <td>Trans-activating enhancer, that can be used to simulate enhancer hijacking</td> |
</tr> | </tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td> | ||
− | <td>mCherry Expression Cassette: UAS, minimal | + | <td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td> |
− | <td>Readout system for enhancer binding | + | <td>Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker</td> |
− | + | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td> | ||
− | <td>Oct1 - 5x UAS | + | <td>Oct1 - 5x UAS Binding Casette</td> |
− | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay | + | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td> | <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td> | ||
− | <td>TRE-minimal | + | <td>TRE-minimal Promoter- Firefly Luciferase</td> |
− | <td>Contains | + | <td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence |
− | simulated enhancer hijacking | + | readout for |
+ | simulated enhancer hijacking</td> | ||
</tr> | </tr> | ||
</tbody> | </tbody> | ||
</table> | </table> | ||
− | |||
</section> | </section> | ||
<section id="1"> | <section id="1"> | ||
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</html> | </html> | ||
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− | <span class= | + | <span class="h3bb">Sequence and Features</span> |
<partinfo>BBa_K5237017 SequenceAndFeatures</partinfo> | <partinfo>BBa_K5237017 SequenceAndFeatures</partinfo> | ||
<!--################################--> | <!--################################--> | ||
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<html> | <html> | ||
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<section id="2"> | <section id="2"> | ||
<h1>2. Usage and Biology</h1> | <h1>2. Usage and Biology</h1> | ||
<section id="2.1"> | <section id="2.1"> | ||
− | <h1>2.1 TetR DNA | + | <h1>2.1 TetR DNA Binding Protein</h1> |
<p> | <p> | ||
The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the | The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the | ||
resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression | resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression | ||
of <i>tetA</i>, which encodes an efflux pump responsible for removing tetracycline from the cell. | of <i>tetA</i>, which encodes an efflux pump responsible for removing tetracycline from the cell. | ||
− | TetR binds selectively to two plaindromic recognition sequences (<i>tetO</i> | + | TetR binds selectively to two plaindromic recognition sequences (<i>tetO</i>>1,2) with high affinity. For DNA |
− | binding to occur tetR adopts a homodimeric structure and binds with two | + | binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs |
(HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a | (HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a | ||
conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth <i>et al.</i> | conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth <i>et al.</i> | ||
2000; Kisker <i>et al.</i> 1995). | 2000; Kisker <i>et al.</i> 1995). | ||
− | <br> | + | <br /> |
Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in | Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in | ||
synthetic | synthetic | ||
biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it | biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it | ||
− | an essential element in the development of gene regulation systems (Berens & Hillen, 2004). | + | an essential element in the development of gene regulation systems (Berens & Hillen, 2004). |
− | <br> | + | <br /> |
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its | In our project, tetR was integrated into the design of a modular DNA-stapling system because of its | ||
well-characterized behavior, ensuring reliable DNA interactions. | well-characterized behavior, ensuring reliable DNA interactions. | ||
Line 332: | Line 386: | ||
</section> | </section> | ||
<section id="2.2"> | <section id="2.2"> | ||
− | <h1>2. | + | <h1>2.2 mScarlet-I</h1> |
<p> | <p> | ||
− | mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels <i>et al.</i> in | + | mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels <i>et al.</i> in 2017. It |
is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its | is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its | ||
maturation | maturation | ||
− | speed and fluorescence properties (Bindels <i>et al.</i>, | + | speed and fluorescence properties (Bindels <i>et al.</i>, 201). mScarlet-I absorbs at 569 nm and emits at 594 nm. |
</p> | </p> | ||
<p> | <p> | ||
Line 343: | Line 397: | ||
mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime | mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime | ||
(3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence | (3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence | ||
− | intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock <i>et al.</i> 2020). When paired with mNeonGreen, mScarlet-I | + | intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock <i>et al.</i> |
+ | 2020). When paired with mNeonGreen, mScarlet-I | ||
+ | </p> | ||
</p> | </p> | ||
</section> | </section> | ||
<section id="2.3"> | <section id="2.3"> | ||
− | + | <h1>2.3 Förster Resonance Energy Transfer (FRET)</h1> | |
− | + | <p>Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred | |
− | + | non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The | |
− | + | efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in | |
− | + | the | |
− | + | range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter <i>et al.</i>, 2019). This | |
− | + | proximity | |
− | + | sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs. | |
− | + | Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's | |
− | + | absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & | |
− | + | Brand, | |
− | + | 1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in | |
− | + | real time. | |
− | + | For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong | |
− | + | fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels | |
− | + | <i>et al.</i>, 2017; Shaner <i>et al.</i>, 2013). FRET's sensitivity to small changes in distance makes it | |
− | + | especially powerful | |
− | + | for analyzing molecular interactions in living cells (Okamoto & Sako, 2017). | |
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</p> | </p> | ||
<div class="thumb"> | <div class="thumb"> | ||
− | <div class="thumbinner" style="width: | + | <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" |
− | src="https://static.igem.wiki/teams/5237/wetlab-results/ | + | src="https://static.igem.wiki/teams/5237/wetlab-results/fpbase-fret-mng-msci.svg" style="width:99%;" /> |
− | + | ||
<div class="thumbcaption"> | <div class="thumbcaption"> | ||
− | <i><b>Figure | + | <i><b>Figure 2: Overview of Excitation and Emission Spectrum of mNeonGreen and m-Scarlet and it's Properties |
− | + | as a FRET Pair</b></i> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</section> | </section> | ||
− | <section id="5"> | + | </section> |
− | <h1>5. | + | <section id="3"> |
− | <p> | + | <h1>3. Assembly and Part Evolution</h1> |
− | for in | + | <p> |
− | + | As part of our engineering efforts an engineered tetR-mScarlet was tested, that binds DNA as a monomer. This scTetR | |
− | </ | + | is a fusion of |
− | </ | + | two tetR proteins with a flexible linker which was described to maintain the same DNA-binding affinity and |
+ | specificity as wild-type tetR (Kaminoka <i>et al</i>. 2006; Krueger <i>et al.</i>2003)</p> | ||
+ | <p> | ||
+ | Our experiments with the scTetR-mScarlet-I fusion protein showed no significant expression. Since the | ||
+ | scTetR-mScarlet-I fusion protein was expressed as part of a polycistronic transcript, the results should be taken | ||
+ | with caution. | ||
+ | Nonetheless we belive that qualitative assesements can still be drawn, because the same construct with | ||
+ | tetR-mScarlet-I showed a good expression (Figure 2, 3). | ||
+ | </p> | ||
+ | <div class="thumb"></div> | ||
+ | <div class="thumbinner" style="width:300px"><img alt="pic" class="thumbimage" | ||
+ | src="https://static.igem.wiki/teams/5237/engineering/sist-mut-t7-mscarlet-i.svg" style="width:99%;" /> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 3: Fluoresence Measurement of scTetR with Mutant T7 Polymerase.</b> Fluorescence intensity of | ||
+ | mScarlet-I (ex. 560 nm, em. 600 nm) was measured 18 h after induction with IPTG and normalized to cell count | ||
+ | (OD<sub>600</sub>). | ||
+ | Positive control consists of a tetR-mNeonGreen fusion protein.</i> | ||
+ | </div> | ||
+ | </div> | ||
+ | </section> | ||
+ | <section id="4"> | ||
+ | <h1>4. Results</h1> | ||
+ | <p> | ||
+ | The FRET assay was developed using a two-plasmid system in bacterial cells. The expression plasmid | ||
+ | contains a tetR binding site (<a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a>) | ||
+ | and expresses three key proteins under the control of a single T7 | ||
+ | promoter in a polycistronic operon: (1) tetR-Oct1, our simple staple fusion protein that acts as a | ||
+ | bivalent DNA-binding protein, tethering two plasmids via tetR and Oct1 binding sites; (2) | ||
+ | Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This | ||
+ | ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains | ||
+ | an Oct1 binding site (<a href="https://parts.igem.org/Part:BBa_K5237018">BBa_K5237018</a>) for the staple and FRET | ||
+ | donor binding. | ||
+ | <br /><br /> | ||
+ | When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I | ||
+ | into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the | ||
+ | plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected | ||
+ | by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control, | ||
+ | consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and | ||
+ | serves as a benchmark for the assay. | ||
+ | </p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:60%;"> | ||
+ | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/figures-corrected/basic-staple-fret.svg" | ||
+ | style="width:99%;" /> | ||
+ | <div class="thumbcaption"> | ||
+ | <i> | ||
+ | <b>Figure 4: Overview FRET Assay</b> | ||
+ | </i> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h | ||
+ | after induction with varying IPTG concentration (Fig. 5). An increasing | ||
+ | expression strength | ||
+ | is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was | ||
+ | significantly stronger compared to the negative control and staple. The negative control and | ||
+ | staple, which both have the same expression plasmid construct, had similar fluorescence intensity | ||
+ | for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in | ||
+ | strong discrepancies. To ensure comparability between the negative control and staple, further | ||
+ | fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. Fluorescence | ||
+ | measurement of the donor and acceptor showed similar intensities, with only a small significant | ||
+ | difference for mNeonGreen. A large difference could be measured between the staple and negative | ||
+ | control, indicating proximity induced FRET. (Fig. 5) | ||
+ | </p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:700px"><img alt="pic" class="thumbimage" | ||
+ | src="https://static.igem.wiki/teams/5237/wetlab-results/sist-results-panel-fret.svg" style="width:99%;" /> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 5: Fluorescence of mNeonGreen, mScarlet-I and FRET.</b> | ||
+ | Fluorescence intensity of mNeonGreen (ex. 490 nm, em. 530 nm), | ||
+ | mScarlet-I (ex. 560 nm, em. 600 nm), and FRET (ex. 490 nm, em. 600 nm) was measured 18 h after induction with | ||
+ | IPTG and normalized to cell count (OD<sub>600</sub>). | ||
+ | <b>A), B)</b> Fluorescence intensity of mNeonGreen and mScarlet-I after induction with different IPTG | ||
+ | concentrations. | ||
+ | <b>C)</b> Fluorescence intensity of FRET pair induced with 0.025 mM IPTG. Statistical significance was | ||
+ | tested for with Ordinary two-way ANOVA with Šidák's multiple comparison test, with a single | ||
+ | pooled variance. *p < 0.05, ****p < 0.001. Only significant results, within groups are shown. Data is | ||
+ | depcited as mean ± SD. | ||
+ | </i> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </section> | ||
+ | <section id="5"> | ||
+ | <h1>5. Conclusion</h1> | ||
+ | <p> | ||
+ | Our FRET assay successfully demonstrated the proximity of two DNA strands in living cells using mNeonGreen and | ||
+ | mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a | ||
+ | positive control. The results showed a significant difference in fluorescence intensity between the staple and | ||
+ | negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and | ||
+ | test out novel Staples. | ||
+ | Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions. | ||
+ | </p> | ||
+ | </section> | ||
+ | <section id="6"> | ||
+ | <h1>6. References</h1> | ||
+ | <p>Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), <em>Genetic | ||
+ | Engineering: Principles and Methods</em> (pp. 255–277). Springer US. <a | ||
+ | href="https://doi.org/10.1007/978-0-306-48573-2_13" | ||
+ | target="_blank">https://doi.org/10.1007/978-0-306-48573-2_13</a></p> | ||
+ | <p>Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G., | ||
+ | Royant, A., Hink, M. A., & Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for | ||
+ | cellular imaging. <em>Nature Methods, 14</em>(1), 53–56. <a href="https://doi.org/10.1038/nmeth.4074" | ||
+ | target="_blank">https://doi.org/10.1038/nmeth.4074</a></p> | ||
+ | <p>Hochreiter, B., Kunze, M., Moser, B., & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative | ||
+ | analysis of protein interactions including stoichiometries and relative affinities in living cells. <em>Scientific | ||
+ | Reports, 9</em>(1), 8233. <a href="https://doi.org/10.1038/s41598-019-44650-0" | ||
+ | target="_blank">https://doi.org/10.1038/s41598-019-44650-0</a></p> | ||
+ | <p>Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., & Saenger, W. (1995). The Complex Formed Between Tet Repressor | ||
+ | and Tetracycline-Mg<sup>2+</sup> Reveals Mechanism of Antibiotic Resistance. <em>Journal of Molecular Biology, | ||
+ | 247</em>(2), 260–280. <a href="https://doi.org/10.1006/jmbi.1994.0138" | ||
+ | target="_blank">https://doi.org/10.1006/jmbi.1994.0138</a></p> | ||
+ | <p>McCullock, T. W., MacLean, D. M., & Kammermeier, P. J. (2020). Comparing the performance of mScarlet-I, mRuby3, | ||
+ | and mCherry as FRET acceptors for mNeonGreen. <em>PLOS ONE, 15</em>(2), e0219886. <a | ||
+ | href="https://doi.org/10.1371/journal.pone.0219886" | ||
+ | target="_blank">https://doi.org/10.1371/journal.pone.0219886</a></p> | ||
+ | <p>Okamoto, K., & Sako, Y. (2017). Recent advances in FRET for the study of protein interactions and dynamics. | ||
+ | <em>Current Opinion in Structural Biology, 46</em>, 16–23. <a href="https://doi.org/10.1016/j.sbi.2017.03.010" | ||
+ | target="_blank">https://doi.org/10.1016/j.sbi.2017.03.010</a></p> | ||
+ | <p>Orth, P., Schnappinger, D., Hillen, W., Saenger, W., & Hinrichs, W. (2000). Structural basis of gene regulation | ||
+ | by the tetracycline inducible Tet repressor-operator system. <em>Nature Structural Biology, 7</em>(3), 215–219. <a | ||
+ | href="https://doi.org/10.1038/73324" target="_blank">https://doi.org/10.1038/73324</a></p> | ||
+ | <p>Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, | ||
+ | R. N., Israelsson, M., Davidson, M. W., & Wang, J. (2013). A bright monomeric green fluorescent protein derived | ||
+ | from <em>Branchiostoma lanceolatum</em>. <em>Nature Methods, 10</em>(5), 407–409. <a | ||
+ | href="https://doi.org/10.1038/nmeth.2413" target="_blank">https://doi.org/10.1038/nmeth.2413</a></p> | ||
+ | <p>Wu, P. G., & Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. <em>Analytical | ||
+ | Biochemistry, 218</em>(1), 1-13. <a href="https://doi.org/10.1006/abio.1994.1134" | ||
+ | target="_blank">https://doi.org/10.1006/abio.1994.1134</a></p> | ||
+ | </section> | ||
</html> | </html> |
Latest revision as of 13:19, 2 October 2024
FRET-Acceptor: TetR-mScarlet-I
This composite part is a fusion protein of TetR and the red fluorescent protein mScarlet-I. It was used as a FRET acceptor in combination with mNeonGreen-Oct1 as the donor. This part was used to measure the proximity of two DNA strands by FRET fluorescence measurements.
Contents
While synthetic biology has in the past focused on engineering the genomic sequence of organisms, the 3D
spatial organization of DNA is well-known to be an important layer of information encoding in
particular in eukaryotes, playing a crucial role in
gene regulation and hence
cell fate, disease development, evolution, and more. However, tools to precisely manipulate and control the
genomic spatial
architecture are limited, hampering the exploration of
3D genome engineering in synthetic biology. We - the iGEM Team Heidelberg 2024 - have developed PICasSO, a
powerful
molecular toolbox for rationally engineering genome 3D architectures in living cells, based on
various DNA-binding proteins.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using engineered "protein staples" in living cells. This enables researchers to recreate naturally occurring alterations of 3D genomic interactions, such as enhancer hijacking in cancer, or to design entirely new spatial architectures for artificial gene regulation and cell function control. Specifically, the fusion of two DNA binding proteins enables to artificially bring otherwise distant genomic loci into spatial proximity. To unlock the system's full potential, we introduce versatile chimeric CRISPR/Cas complexes, connected either at the protein or - in the case of CRISPR/Cas-based DNA binding moieties - the guide RNA level. These complexes are referred to as protein- or Cas staples, respectively. Beyond its versatility with regard to the staple constructs themselves, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples in vitro and in vivo. Notably, the PICasSO toolbox was developed in a design-build-test-learn engineering cycle closely intertwining wet lab experiments and computational modeling and iterated several times, yielding a collection of well-functioning and -characterized parts.
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding
proteins
include our
finalized Cas staple experimentally validated using an artificial "enhancer hijacking" system as well as
"half staples" that can be combined by scientists to compose entirely
new Cas staples in the future. We also include our Simple staples comprised of particularly small, simple
and robust DNA binding domains well-known to the synthetic biology community, which serve as controls for
successful stapling
and can be further engineered to create alternative, simpler, and more compact staples.
(ii) As functional elements, we list additional parts that enhance and expand the
functionality of our Cas and
Basic staples. These
consist of staples dependent on
cleavable peptide linkers targeted by cancer-specific proteases or inteins that allow condition-specific,
dynamic stapling in vivo.
We also include several engineered parts that enable the efficient delivery of PICasSO's constructs into
target cells, including mammalian cells,
with our new
interkingdom conjugation system.
(iii) As the final category of our collection, we provide parts that underlie our custom
readout
systems. These include components of our established FRET-based proximity assay system, enabling
users to
confirm
accurate stapling. Additionally, we offer a complementary, application-oriented testing system based on a
luciferase reporter, which allows for straightforward experimental assessment of functional enhancer
hijacking events
in mammalian cells.
The following table gives a comprehensive overview of all parts in our PICasSO toolbox. The highlighted parts showed
exceptional performance as described on our iGEM wiki and can serve as a reference. The other
parts in
the
collection are versatile building blocks designed to provide future iGEMers with the flexibility to engineer
their
own custom Cas staples, enabling further optimization and innovation in the new field of 3D genome
engineering.
Our part collection includes:
DNA-Binding Proteins: Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions in vivo | ||
BBa_K5237000 | Fusion Guide RNA Entry Vector MbCas12a-SpCas9 | Entry vector for simple fgRNA cloning via SapI |
BBa_K5237001 | Staple Subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple |
BBa_K5237002 | Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple |
BBa_K5237003 | Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into close proximity |
BBa_K5237004 | Staple Subunit: Oct1-DBD | Staple subunit that can be combined to form a functional staple, for example with TetR. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
BBa_K5237005 | Staple Subunit: TetR | Staple subunit that can be combined to form a functional staple, for example with Oct1. Can also be combined with a fluorescent protein as part of the FRET proximity assay |
BBa_K5237006 | Simple Staple: TetR-Oct1 | Functional staple that can be used to bring two DNA strands in close proximity |
BBa_K5237007 | Staple Subunit: GCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
BBa_K5237008 | Staple Subunit: rGCN4 | Staple subunit that can be combined to form a functional staple, for example with rGCN4 |
BBa_K5237009 | Mini Staple: bGCN4 | Assembled staple with minimal size that can be further engineered | Functional Elements: Protease-cleavable peptide linkers and inteins are used to control and modify staples for further optimization for custom applications |
BBa_K5237010 | Cathepsin B-cleavable Linker: GFLG | Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make responsive staples |
BBa_K5237011 | Cathepsin B Expression Cassette | Expression cassette for the overexpression of cathepsin B |
BBa_K5237012 | Caged NpuN Intein | A caged NpuN split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
BBa_K5237013 | Caged NpuC Intein | A caged NpuC split intein fragment that undergoes protein trans-splicing after protease activation, which can be used to create functionalized staple subunits |
BBa_K5237014 | Fusion Guide RNA Processing Casette | Processing cassette to produce multiple fgRNAs from one transcript, that can be used for multiplexed 3D genome reprogramming |
BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for large constructs |
BBa_K4643003 | IncP Origin of Transfer | Origin of transfer that can be cloned into the plasmid vector and used for conjugation as a means of delivery | Readout Systems: FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and mammalian cells |
BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | FRET donor-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to visualize DNA-DNA proximity |
BBa_K5237017 | FRET-Acceptor: TetR-mScarlet-I | Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize DNA-DNA proximity |
BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET proximity assay |
BBa_K5237019 | TetR Binding Cassette | DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the FRET proximity assay | BBa_K5237020 | Cathepsin B-Cleavable Trans-Activator: NLS-Gal4-GFLG-VP64 | Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker |
BBa_K5237021 | NLS-Gal4-VP64 | Trans-activating enhancer, that can be used to simulate enhancer hijacking | BBa_K5237022 | mCherry Expression Cassette: UAS, minimal Promoter, mCherry | Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker |
BBa_K5237023 | Oct1 - 5x UAS Binding Casette | Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay |
BBa_K5237024 | TRE-minimal Promoter- Firefly Luciferase | Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence readout for simulated enhancer hijacking |
1. Sequence overview
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1149
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 623
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 466
The tetracycline repressor protein (tetR) is naturally present in gram-negative bacteria and is involved in the
resistance mechanism against tetracycline (and derivatives). It does so by tightly controlling the gene expression
of tetA, which encodes an efflux pump responsible for removing tetracycline from the cell.
TetR binds selectively to two plaindromic recognition sequences (tetO>1,2) with high affinity. For DNA
binding to occur tetR adopts a homodimeric structure and binds with two α-helix-turn- α-helix motifs
(HTH) to two tandemly oriented tetO sequences. In the presence of tetracycline or its analogs, tetR undergoes a
conformational change, which prevents it from binding to DNA, therby allowing gene expression(Orth et al.
2000; Kisker et al. 1995).
mScarlet-I is a rapidly-maturing monomeric red fluorescent protein designed by Bindels et al. in 2017. It
is a derivative of the original mScarlet, with a single amino acid substitution (T74I) that enhances its
maturation
speed and fluorescence properties (Bindels et al., 201). mScarlet-I absorbs at 569 nm and emits at 594 nm.
mScarlet-I stands out among red fluorescent proteins for its high quantum yield (0.54), long fluorescence lifetime
(3.1 ns), and rapid maturation time of approximately 36 minutes. These features ensure strong fluorescence
intensity and make it an ideal candidate for Förster Resonance Energy Transfer assays (McCullock et al.
2020). When paired with mNeonGreen, mScarlet-I
2. Usage and Biology
2.1 TetR DNA Binding Protein
Due to its robust and highly regulatable DNA-binding properties, tetR has become a widely adopted tool in
synthetic
biology. Its ease of modification and ability to function in both prokaryotic and eukaryotic systems have made it
an essential element in the development of gene regulation systems (Berens & Hillen, 2004).
In our project, tetR was integrated into the design of a modular DNA-stapling system because of its
well-characterized behavior, ensuring reliable DNA interactions.
2.2 mScarlet-I
2.3 Förster Resonance Energy Transfer (FRET)
Förster Resonance Energy Transfer (FRET) is a distance-dependent physical process where energy is transferred non-radiatively from an excited donor fluorophore to an acceptor fluorophore via dipole-dipole coupling. The efficiency of energy transfer is highly sensitive to the distance between the donor and acceptor, typically in the range of 1-10 nm, making FRET ideal for studying molecular proximity (Hochreiter et al., 2019). This proximity sensitivity is governed by the Förster radius (R₀), which is the distance at which 50% energy transfer occurs. Factors affecting FRET efficiency include the overlap of the donor's emission spectrum with the acceptor's absorption spectrum, the quantum yield of the donor, and the relative orientation of the fluorophores (Wu & Brand, 1994). These characteristics allow FRET to detect interactions such as protein-DNA binding or DNA proximity in real time. For our assay, we selected mNeonGreen and mScarlet-I as donor and acceptor, respectively, due to their strong fluorescence, spectral overlap, and minimal photobleaching, ensuring high FRET efficiency in our system (Bindels et al., 2017; Shaner et al., 2013). FRET's sensitivity to small changes in distance makes it especially powerful for analyzing molecular interactions in living cells (Okamoto & Sako, 2017).
3. Assembly and Part Evolution
As part of our engineering efforts an engineered tetR-mScarlet was tested, that binds DNA as a monomer. This scTetR is a fusion of two tetR proteins with a flexible linker which was described to maintain the same DNA-binding affinity and specificity as wild-type tetR (Kaminoka et al. 2006; Krueger et al.2003)
Our experiments with the scTetR-mScarlet-I fusion protein showed no significant expression. Since the scTetR-mScarlet-I fusion protein was expressed as part of a polycistronic transcript, the results should be taken with caution. Nonetheless we belive that qualitative assesements can still be drawn, because the same construct with tetR-mScarlet-I showed a good expression (Figure 2, 3).
4. Results
The FRET assay was developed using a two-plasmid system in bacterial cells. The expression plasmid
contains a tetR binding site (BBa_K5237019)
and expresses three key proteins under the control of a single T7
promoter in a polycistronic operon: (1) tetR-Oct1, our simple staple fusion protein that acts as a
bivalent DNA-binding protein, tethering two plasmids via tetR and Oct1 binding sites; (2)
Oct1-mNeonGreen, serving as the FRET donor; and (3) tetR-mScarlet-I, the FRET acceptor. This
ensures all three proteins are co-expressed in similar stoichiometry. The folding plasmid contains
an Oct1 binding site (BBa_K5237018) for the staple and FRET
donor binding.
When tetR-Oct1 binds its respective sites on both plasmids, it brings mNeonGreen and mScarlet-I
into close proximity, facilitating FRET between the two fluorophores. Successful stapling of the
plasmids results in increased energy transfer from mNeonGreen to mScarlet-I, which can be detected
by exciting mNeonGreen and measuring enhanced emission from mScarlet-I. A positive control,
consisting of a direct fusion of mNeonGreen and mScarlet-I, ensures maximal FRET efficiency and
serves as a benchmark for the assay.
Fluorescence intensity, normalized to cell count, of mNeonGreen and mScarlet-I was measured 18 h after induction with varying IPTG concentration (Fig. 5). An increasing expression strength is visible for decreasing IPTG concentrations. Fluorescence intensity of the positive control was significantly stronger compared to the negative control and staple. The negative control and staple, which both have the same expression plasmid construct, had similar fluorescence intensity for mNeonGreen and mScarlet-I down to approximately 0.05 mM. Lower concentrations resulted in strong discrepancies. To ensure comparability between the negative control and staple, further fluorescence intensity measurements were conducted after induction with 0.05 mM IPTG. Fluorescence measurement of the donor and acceptor showed similar intensities, with only a small significant difference for mNeonGreen. A large difference could be measured between the staple and negative control, indicating proximity induced FRET. (Fig. 5)
5. Conclusion
Our FRET assay successfully demonstrated the proximity of two DNA strands in living cells using mNeonGreen and mScarlet-I as a donor-acceptor pair. The assay was optimized for maximal FRET efficiency and validated with a positive control. The results showed a significant difference in fluorescence intensity between the staple and negative control, indicating successful DNA stapling and FRET. This assay provides a powerful tool to engineer and test out novel Staples. Future work will focus on further optimizing the assay in mammalian cells and quantifying interactions.
6. References
Berens, C., & Hillen, W. (2004). Gene Regulation By Tetracyclines. In J. K. Setlow (Ed.), Genetic Engineering: Principles and Methods (pp. 255–277). Springer US. https://doi.org/10.1007/978-0-306-48573-2_13
Bindels, D. S., Haarbosch, L., van Weeren, L., Postma, M., Wiese, K. E., Mastop, M., Aumonier, S., Gotthard, G., Royant, A., Hink, M. A., & Gadella, T. W. J. (2017). mScarlet: A bright monomeric red fluorescent protein for cellular imaging. Nature Methods, 14(1), 53–56. https://doi.org/10.1038/nmeth.4074
Hochreiter, B., Kunze, M., Moser, B., & Schmid, J. A. (2019). Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells. Scientific Reports, 9(1), 8233. https://doi.org/10.1038/s41598-019-44650-0
Kisker, C., Hinrichs, W., Tovar, K., Hillen, W., & Saenger, W. (1995). The Complex Formed Between Tet Repressor and Tetracycline-Mg2+ Reveals Mechanism of Antibiotic Resistance. Journal of Molecular Biology, 247(2), 260–280. https://doi.org/10.1006/jmbi.1994.0138
McCullock, T. W., MacLean, D. M., & Kammermeier, P. J. (2020). Comparing the performance of mScarlet-I, mRuby3, and mCherry as FRET acceptors for mNeonGreen. PLOS ONE, 15(2), e0219886. https://doi.org/10.1371/journal.pone.0219886
Okamoto, K., & Sako, Y. (2017). Recent advances in FRET for the study of protein interactions and dynamics. Current Opinion in Structural Biology, 46, 16–23. https://doi.org/10.1016/j.sbi.2017.03.010
Orth, P., Schnappinger, D., Hillen, W., Saenger, W., & Hinrichs, W. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature Structural Biology, 7(3), 215–219. https://doi.org/10.1038/73324
Shaner, N. C., Lambert, G. G., Chammas, A., Ni, Y., Cranfill, P. J., Baird, M. A., Sell, B. R., Allen, J. R., Day, R. N., Israelsson, M., Davidson, M. W., & Wang, J. (2013). A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nature Methods, 10(5), 407–409. https://doi.org/10.1038/nmeth.2413
Wu, P. G., & Brand, L. (1994). Resonance Energy Transfer: Methods and Applications. Analytical Biochemistry, 218(1), 1-13. https://doi.org/10.1006/abio.1994.1134