Difference between revisions of "Part:BBa K5237004"
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− | + | <!-- Part summary --> | |
− | + | <section> | |
− | + | <h1>Half staple: Oct1-DBD</h1> | |
− | + | <p>Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor. It can be readily fused with other | |
− | + | DNA-binding proteins to form a functional staple to DNA-DNA proximity. We used this part as a component for our | |
− | + | Simple | |
− | + | staple (<a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a>) and also fused to | |
− | + | mNeonGreen, as part of a FRET readout system (<a href="https://parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a>). | |
− | + | </p> | |
− | + | <p></p> | |
− | + | </section> | |
− | + | <div class="toc" id="toc"> | |
− | + | <div id="toctitle"> | |
− | + | <h1>Contents</h1> | |
− | + | </div> | |
− | + | <ul> | |
− | + | <li class="toclevel-1 tocsection-1"><a href="#1"><span class="tocnumber">1</span> <span class="toctext">Sequence | |
+ | Overview</span></a> | ||
+ | </li> | ||
+ | <li class="toclevel-1 tocsection-2"><a href="#2"><span class="tocnumber">2</span> <span class="toctext">Usage and | ||
Biology</span></a> | Biology</span></a> | ||
− | + | </li> | |
− | + | <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 class="toclevel-1 tocsection-5"><a href="#4"><span class="tocnumber">4</span> <span class="toctext">Results</span></a> | |
− | + | <ul> | |
− | + | <li class="toclevel-2 tocsection-4"><a href="#4.1"><span class="tocnumber">4.1</span> <span class="toctext">Protein Expression and EMSA</span></a> | |
− | + | </li> | |
− | + | <li class="toclevel-2 tocsection-5"><a href="#4.2"><span class="tocnumber">4.2</span> <span class="toctext"><i>In Silico</i> Characterization Using DaVinci</span></a> | |
− | + | </li> | |
− | + | </ul> | |
− | + | </li> | |
− | + | <li class="toclevel-1 tocsection-8"><a href="#5"><span class="tocnumber">5</span> <span class="toctext">References</span></a> | |
− | <h1>1. Sequence overview</h1> | + | </li> |
− | + | </ul> | |
+ | </div> | ||
+ | <section><p><br/><br/></p> | ||
+ | <font size="5"><b>The PICasSO Toolbox </b> </font> | ||
+ | <div class="thumb" style="margin-top:10px;"></div> | ||
+ | <div class="thumbinner" style="width:550px"><img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/registry-part-collection-engineering-cycle-example-overview.svg" style="width:99%;"/> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 1: How our part collection can be used to engineer new staples</b></i> | ||
+ | </div> | ||
+ | </div> | ||
+ | <p> | ||
+ | <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 | ||
+ | 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 | ||
+ | <b>powerful | ||
+ | molecular toolbox for rationally engineering genome 3D architectures</b> in living cells, based on | ||
+ | various DNA-binding proteins. | ||
+ | </p> | ||
+ | <p> | ||
+ | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | ||
+ | <b>re-programming | ||
+ | of DNA-DNA interactions</b> 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 <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>At its heart, the PICasSO part collection consists of three categories. <br/><b>(i)</b> Our <b>DNA-binding | ||
+ | proteins</b> | ||
+ | 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. <br/> | ||
+ | <b>(ii)</b> As <b>functional elements</b>, 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 <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 | ||
+ | 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> | ||
+ | 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 | ||
+ | exceptional performance as described on our iGEM wiki and can serve as a reference.</mark> 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.<br/> | ||
+ | </p> | ||
+ | <p> | ||
+ | <font size="4"><b>Our part collection includes:</b></font><br/> | ||
+ | </p> | ||
+ | <table style="width: 90%; padding-right:10px;"> | ||
+ | <td align="left" colspan="3"><b>DNA-Binding Proteins: </b> | ||
+ | Modular building blocks for engineering of custom staples to mediate defined DNA-DNA interactions <i>in vivo</i></td> | ||
+ | <tbody> | ||
+ | <tr bgcolor="#FFD700"> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | ||
+ | <td>Fusion Guide RNA Entry Vector MbCas12a-SpCas9</td> | ||
+ | <td>Entry vector for simple fgRNA cloning via SapI</td> | ||
+ | </tr> | ||
+ | <tr bgcolor="#FFD700"> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237001" target="_blank">BBa_K5237001</a></td> | ||
+ | <td>Staple Subunit: dMbCas12a-Nucleoplasmin NLS</td> | ||
+ | <td>Staple subunit that can be combined with crRNA or fgRNA and dSpCas9 to form a functional staple | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr bgcolor="#FFD700"> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a></td> | ||
+ | <td>Staple Subunit: SV40 NLS-dSpCas9-SV40 NLS</td> | ||
+ | <td>Staple subunit that can be combined with a sgRNA or fgRNA and dMbCas12a to form a functional staple | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237003" target="_blank">BBa_K5237003</a></td> | ||
+ | <td>Cas Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS</td> | ||
+ | <td>Functional Cas staple that can be combined with sgRNA and crRNA or fgRNA to bring two DNA strands into | ||
+ | close | ||
+ | proximity | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237004" target="_blank">BBa_K5237004</a></td> | ||
+ | <td>Staple Subunit: Oct1-DBD</td> | ||
+ | <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> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237005" target="_blank">BBa_K5237005</a></td> | ||
+ | <td>Staple Subunit: TetR</td> | ||
+ | <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> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237006" target="_blank">BBa_K5237006</a></td> | ||
+ | <td>Simple Staple: TetR-Oct1</td> | ||
+ | <td>Functional staple that can be used to bring two DNA strands in close proximity</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237007" target="_blank">BBa_K5237007</a></td> | ||
+ | <td>Staple Subunit: GCN4</td> | ||
+ | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237008" target="_blank">BBa_K5237008</a></td> | ||
+ | <td>Staple Subunit: rGCN4</td> | ||
+ | <td>Staple subunit that can be combined to form a functional staple, for example with rGCN4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237009" target="_blank">BBa_K5237009</a></td> | ||
+ | <td>Mini Staple: bGCN4</td> | ||
+ | <td> | ||
+ | Assembled staple with minimal size that can be further engineered</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | <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 | ||
+ | for custom applications</td> | ||
+ | <tbody> | ||
+ | <tr bgcolor="#FFD700"> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237010" target="_blank">BBa_K5237010</a></td> | ||
+ | <td>Cathepsin B-cleavable Linker: GFLG</td> | ||
+ | <td>Cathepsin B-cleavable peptide linker that can be used to combine two staple subunits to make | ||
+ | responsive | ||
+ | staples</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237011" target="_blank">BBa_K5237011</a></td> | ||
+ | <td>Cathepsin B Expression Cassette</td> | ||
+ | <td>Expression cassette for the overexpression of cathepsin B</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237012" target="_blank">BBa_K5237012</a></td> | ||
+ | <td>Caged NpuN Intein</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> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237013" target="_blank">BBa_K5237013</a></td> | ||
+ | <td>Caged NpuC Intein</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> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> | ||
+ | <td>Fusion Guide RNA Processing Casette</td> | ||
+ | <td>Processing cassette to produce multiple fgRNAs from one transcript, that can be used for | ||
+ | multiplexed 3D | ||
+ | genome reprogramming</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237015" target="_blank">BBa_K5237015</a></td> | ||
+ | <td>Intimin anti-EGFR Nanobody</td> | ||
+ | <td>Interkingdom conjugation between bacteria and mammalian cells, as an alternative delivery tool for | ||
+ | large | ||
+ | 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> | ||
+ | </tbody> | ||
+ | <td align="left" colspan="3"><b>Readout Systems: </b> | ||
+ | FRET and enhancer recruitment readout systems to rapidly assess successful DNA stapling in bacterial and | ||
+ | mammalian cells | ||
+ | </td> | ||
+ | <tbody> | ||
+ | <tr bgcolor="#FFD700"> | ||
+ | <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-fluorophore fused to Oct1-DBD that binds to the Oct1 binding cassette, which can be used to | ||
+ | visualize | ||
+ | DNA-DNA | ||
+ | proximity</td> | ||
+ | </tr> | ||
+ | <tr bgcolor="#FFD700"> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237017" target="_blank">BBa_K5237017</a></td> | ||
+ | <td>FRET-Acceptor: TetR-mScarlet-I</td> | ||
+ | <td>Acceptor part for the FRET assay binding the TetR binding cassette, which can be used to visualize | ||
+ | DNA-DNA | ||
+ | proximity</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237018" target="_blank">BBa_K5237018</a></td> | ||
+ | <td>Oct1 Binding Casette</td> | ||
+ | <td>DNA sequence containing 12 Oct1 binding motifs, compatible with various assays such as the FRET | ||
+ | proximity assay</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237019" target="_blank">BBa_K5237019</a></td> | ||
+ | <td>TetR Binding Cassette</td> | ||
+ | <td>DNA sequence containing 12 Oct1 binding motifs, can be used for different assays such as the | ||
+ | FRET | ||
+ | proximity assay</td> | ||
+ | </tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237020" target="_blank">BBa_K5237020</a></td> | ||
+ | <td>Cathepsin B-Cleavable <i>Trans</i>-Activator: NLS-Gal4-GFLG-VP64</td> | ||
+ | <td>Readout system that responds to protease activity, which was used to test cathepsin B-cleavable linker | ||
+ | </td> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237021" target="_blank">BBa_K5237021</a></td> | ||
+ | <td>NLS-Gal4-VP64</td> | ||
+ | <td><i>Trans</i>-activating enhancer, that can be used to simulate enhancer hijacking</td> | ||
+ | </tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237022" target="_blank">BBa_K5237022</a></td> | ||
+ | <td>mCherry Expression Cassette: UAS, minimal Promoter, mCherry</td> | ||
+ | <td>Readout system for enhancer binding, which was used to test cathepsin B-cleavable linker</td> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237023" target="_blank">BBa_K5237023</a></td> | ||
+ | <td>Oct1 - 5x UAS Binding Casette</td> | ||
+ | <td>Oct1 and UAS binding cassette, that was used for the simulated enhancer hijacking assay</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><a href="https://parts.igem.org/Part:BBa_K5237024" target="_blank">BBa_K5237024</a></td> | ||
+ | <td>TRE-minimal Promoter- Firefly Luciferase</td> | ||
+ | <td>Contains firefly luciferase controlled by a minimal promoter, which was used as a luminescence | ||
+ | readout for | ||
+ | simulated enhancer hijacking</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table></section> | ||
+ | <section id="1"> | ||
+ | <h1>1. Sequence overview</h1> | ||
+ | </section> | ||
</body> | </body> | ||
− | |||
</html> | </html> | ||
− | + | <!--################################--> | |
− | <span class= | + | <span class="h3bb">Sequence and Features</span> |
− | <partinfo> | + | <partinfo>BBa_K5237004 SequenceAndFeatures</partinfo> |
− | + | <!--################################--> | |
− | <html> | + | <html> |
<body> | <body> | ||
− | + | <section id="2"> | |
− | + | <h1>2. Usage and Biology</h1> | |
− | <div class="thumb"> | + | <p>Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in |
− | + | gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the | |
− | + | octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity | |
− | + | (Lundbäck <i>et al.</i>, 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which | |
− | + | work | |
− | + | together to form a stable complex with DNA (Park <i>et al.</i>, 2013, Stepchenko <i>et al.</i> 2021). | |
− | + | </p> | |
− | + | <p>In synthetic biology, Oct1-DBD has been previously used for plasmid display technology due to its strong binding | |
− | + | affinity (K<sub>D</sub> = 9 × 10<sup>-11</sup> M), additionally proteins fused with Oct1-DBD showed increased | |
− | + | expression | |
− | + | and protein solubility (Parker <i>et al.</i> 2020). | |
− | + | </p> | |
− | + | <p> | |
− | + | This part was further used in <a href="https:parts.igem.org/Part:BBa_K5237002" target="_blank">BBa_K5237002</a> as | |
− | + | a fusion with tetR, resulting in a bivalent DNA binding staple, and | |
− | + | also fused to mNeonGreen, as part of a FRET readout system (<a href="https:parts.igem.org/Part:BBa_K5237016" target="_blank">BBa_K5237016</a>). | |
− | + | </p> | |
− | + | </section> | |
− | + | <section id="3"> | |
− | + | <h1>3. Assembly and Part Evolution</h1> | |
− | + | <p>The Oct1-DBD amino acid sequence was obtained from UniProt (<a href="https://www.uniprot.org/uniprot/P14859" target="_blank">P14859</a>, POU domain, class 2, transcription factor 1) | |
− | + | and the DNA binding domain was extracted based on information given from Park <i>et al.</i> 2013 & 2020. | |
− | + | An <i>E. coli</i> codon optimized DNA sequence was obtained through gene synthesis and used to clone further | |
− | + | constructs. | |
− | + | </p> | |
− | + | </section> | |
− | + | <section id="4"> | |
− | + | <h1>4. Results</h1> | |
− | + | <section id="4.1"> | |
− | + | <h2>4.1 Protein Expression and EMSA</h2> | |
− | + | <p>Oct1 was N-terminally fused to His6-mNeonGreen. This fusion protein was expressed under a T7 promoter and | |
− | + | subsequently | |
− | + | purified using metal affinity chromatography with Ni-NTA beads (Fig. 2, left). | |
− | + | DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three | |
− | + | different | |
+ | buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, | ||
+ | 1.8 | ||
+ | mM KH<sub>2</sub>HPO<sub>4</sub>, | ||
+ | 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: | ||
+ | Na<sub>2</sub>HPO<sub>4</sub>, 150 mM | ||
+ | NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Fig. 2, right) | ||
+ | </p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:90%;"> | ||
+ | <div style="display: flex; justify-content: center; border:none;"> | ||
+ | <div> | ||
+ | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/sds-page-mng-oct1-expression.svg" style="height: 300px; width: auto; border:none;"/> | ||
+ | </div> | ||
+ | <div> | ||
+ | <a href="Fig2_right"> | ||
+ | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/wetlab-results/emsa-oct1-binding-buffer-optimization.svg" style="height: 300px; width: auto; border:none;"/> | ||
+ | </a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="thumbcaption" style="text-align: justify;"> | ||
+ | <i><b>Figure 2: Expression and DNA Binding Analysis of His<sub>6</sub>-mNeonGreen-Oct1-DBD.</b></i><br/> | ||
+ | <i><b>Left image:</b>Lane 1: raw lysate of E. coli expression culture after sterile filtration; Lane 2: Flow | ||
+ | through of first wash | ||
+ | (10 bed volumes of NaP10 (Na<sub>2</sub>HPO<sub>4</sub>, 150 mM NaCl, 10 mM Imidazol)); Lane 3: Flow | ||
+ | through | ||
+ | of | ||
+ | second wash (10 bed volumes of NaP20 (Na<sub>2</sub>HPO<sub>4</sub>, 150 mM NaCl, 20 mM Imidazol)); Lane | ||
+ | 4: | ||
+ | Elution of purified protein.<br/> | ||
+ | 1 µL of each fraction was loaded after mixing and heating with 4x Laemmli buffer, on a 4-15% TGX-Gel. The | ||
+ | expected band size of the protein is 56 840.23 Da, highlighted in red on the gel.<br/> | ||
+ | <b>Right image</b> Purified mNeonGreen-Oct1 fusion-protein (1000 nM, 100 nM or 10 nM) were equilibrated | ||
+ | with 0.5 µM DNA, | ||
+ | containing three Oct1 binding sites, in different buffer compositions. | ||
+ | (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 | ||
+ | mM | ||
+ | EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: 50 mM NaH2PO4, 150 mM NaCl, 250 mM Imidazol) Bands | ||
+ | were visualized with SYBR-Safe staining.</i> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </section> | ||
+ | <section id="4.2"> | ||
+ | <h2>4.2 <i>In Silico</i> Characterization using DaVinci</h2> | ||
+ | <p> | ||
+ | We developed the <i>in silico</i> model <a href="https://2024.igem.wiki/heidelberg/model" target="_blank">DaVinci</a> | ||
+ | for rapid engineering | ||
+ | and development of our PICasSO system. | ||
+ | DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system, | ||
+ | refine experimental parameters, and find optimal connections between protein staples and target DNA. | ||
+ | We calibrated DaVinci with literature and our own experimental affinity data calculated from EMSA assays with | ||
+ | purified proteins | ||
+ | DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged | ||
+ | DNA | ||
+ | dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the | ||
+ | DNA-binding interaction.<br/> | ||
+ | The structures shown in Figure 4 were predicted using the AlphaFold server and the protein-DNA interaction | ||
+ | further | ||
+ | analyzed with all atom dynamics simulations. The depicted structures show favorable DNA binding, and no apparent | ||
+ | problems with the fusion protein and DNA binding were detected. | ||
+ | </p> | ||
+ | <div class="thumb"> | ||
+ | <div class="thumbinner" style="width:80%;"> | ||
+ | <img alt="" class="thumbimage" src="https://static.igem.wiki/teams/5237/model/structure-figure-2-png.svg" style="width: 99%;"/> | ||
+ | <div class="thumbcaption"> | ||
+ | <i><b>Figure 4: Representations of the Simple Staple Constructs</b> | ||
+ | Proteins are shown in full color (top row) and by their predicted structural accuracy during DNA | ||
+ | interaction. | ||
+ | The linkers were selected based on their structural property providing maximal flexibility. All structures | ||
+ | were predicted using the AlphaFold server (Google DeepMind, 2024).</i> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div></section> | ||
+ | </section> | ||
+ | <section id="5"> | ||
+ | <h1>5. References</h1> | ||
+ | <p>Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of | ||
+ | Sequence-Specific DNA Binding by the Transcription Factor Oct-1. <em>Biochemistry, 39</em>(25), 7570–7579. <a href="https://doi.org/10.1021/bi000377h" target="_blank">https://doi.org/10.1021/bi000377h</a></p> | ||
+ | <p>Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 | ||
+ | DNA-binding domain suitable for in vitro screening of engineered proteins. <em>Journal of Bioscience and | ||
+ | Bioengineering, 116</em>(2), 246–252. <a href="https://doi.org/10.1016/j.jbiosc.2013.02.005" target="_blank">https://doi.org/10.1016/j.jbiosc.2013.02.005</a></p> | ||
+ | <p>Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., | ||
+ | Kim, S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve | ||
+ | Whole-Cell Biotransformation Efficiency. <em>Frontiers in Bioengineering and Biotechnology, 7</em>. <a href="https://doi.org/10.3389/fbioe.2019.00444" target="_blank">https://doi.org/10.3389/fbioe.2019.00444</a></p> | ||
+ | <p>Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., & | ||
+ | Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal | ||
+ | cells from stress. <em>Scientific Reports, 11</em>(1), 18808. <a href="https://doi.org/10.1038/s41598-021-98323-y" target="_blank">https://doi.org/10.1038/s41598-021-98323-y</a></p> | ||
+ | </section> | ||
</body> | </body> | ||
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Latest revision as of 12:43, 2 October 2024
Half staple: Oct1-DBD
Oct1-DBD is the DNA-binding domain of the human Oct1 transcription factor. It can be readily fused with other DNA-binding proteins to form a functional staple to DNA-DNA proximity. We used this part as a component for our Simple staple (BBa_K5237006) and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).
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]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2. Usage and Biology
Oct1-DBD is the DNA-binding domain of the human transcription factor Oct1 (POU2F1), which plays a key role in gene regulation, immune response, and stress adaptation in eukaryotic cells. This domain specifically binds to the octamer motif (5'-ATGCAAAT-3') within promoter and enhancer regions, influencing transcriptional activity (Lundbäck et al., 2000). The Oct1-DBD consists of both a POU-specific domain and a POU homeodomain, which work together to form a stable complex with DNA (Park et al., 2013, Stepchenko et al. 2021).
In synthetic biology, Oct1-DBD has been previously used for plasmid display technology due to its strong binding affinity (KD = 9 × 10-11 M), additionally proteins fused with Oct1-DBD showed increased expression and protein solubility (Parker et al. 2020).
This part was further used in BBa_K5237002 as a fusion with tetR, resulting in a bivalent DNA binding staple, and also fused to mNeonGreen, as part of a FRET readout system (BBa_K5237016).
3. Assembly and Part Evolution
The Oct1-DBD amino acid sequence was obtained from UniProt (P14859, POU domain, class 2, transcription factor 1) and the DNA binding domain was extracted based on information given from Park et al. 2013 & 2020. An E. coli codon optimized DNA sequence was obtained through gene synthesis and used to clone further constructs.
4. Results
4.1 Protein Expression and EMSA
Oct1 was N-terminally fused to His6-mNeonGreen. This fusion protein was expressed under a T7 promoter and subsequently purified using metal affinity chromatography with Ni-NTA beads (Fig. 2, left). DNA binding affinity was estimated with an electrophoretic mobility shift assay (EMSA). For this, three different buffer conditions were tested (Binding buffer 1: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2HPO4, 0.1 % (v/v) IGEPAL® CA-360, 1 mM EDTA; Binding buffer 2: 10 mM Tris, 50 mM KCl; NaP250: Na2HPO4, 150 mM NaCl, 250 mM Imidazol). DNA binding could only be detected for Binding buffer 1. (Fig. 2, right)
4.2 In Silico Characterization using DaVinci
We developed the in silico model DaVinci
for rapid engineering
and development of our PICasSO system.
DaVinci acts as a digital twin to PICasSO, designed to understand the forces acting on our system,
refine experimental parameters, and find optimal connections between protein staples and target DNA.
We calibrated DaVinci with literature and our own experimental affinity data calculated from EMSA assays with
purified proteins
DaVinci is divided into three phases: static structure prediction, all-atom dynamics simulation, and long-ranged
DNA
dynamics simulation. We applied the first two to our parts, characterizing structure and dynamics of the
DNA-binding interaction.
The structures shown in Figure 4 were predicted using the AlphaFold server and the protein-DNA interaction
further
analyzed with all atom dynamics simulations. The depicted structures show favorable DNA binding, and no apparent
problems with the fusion protein and DNA binding were detected.
5. References
Lundbäck, T., Chang, J.-F., Phillips, K., Luisi, B., & Ladbury, J. E. (2000). Characterization of Sequence-Specific DNA Binding by the Transcription Factor Oct-1. Biochemistry, 39(25), 7570–7579. https://doi.org/10.1021/bi000377h
Park, J. H., Kwon, H. W., & Jeong, K. J. (2013). Development of a plasmid display system with an Oct-1 DNA-binding domain suitable for in vitro screening of engineered proteins. Journal of Bioscience and Bioengineering, 116(2), 246–252. https://doi.org/10.1016/j.jbiosc.2013.02.005
Park, Y., Shin, J., Yang, J., Kim, H., Jung, Y., Oh, H., Kim, Y., Hwang, J., Park, M., Ban, C., Jeong, K. J., Kim, S.-K., & Kweon, D.-H. (2020). Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency. Frontiers in Bioengineering and Biotechnology, 7. https://doi.org/10.3389/fbioe.2019.00444
Stepchenko, A. G., Portseva, T. N., Glukhov, I. A., Kotnova, A. P., Lyanova, B. M., Georgieva, S. G., & Pankratova, E. V. (2021). Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress. Scientific Reports, 11(1), 18808. https://doi.org/10.1038/s41598-021-98323-y