Difference between revisions of "Part:BBa K5237007"
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| − | + | <body> | |
| − | + | <!-- Part summary --> | |
| − | + | <section id="1"> | |
| − | + | <h1>Staple subunit: GCN4</h1> | |
| − | + | <p>GCN4 is a yeast transcription factor belonging to the bZip family of DNA-binding proteins. | |
| − | + | We use GCN4 to study DNA-binding kinetics in our "Mini staples" that bring two DNA target sites into proximity by binding them simultaneously. | |
| − | + | </p> | |
| − | + | <p> </p> | |
| − | + | </section> | |
| − | + | <div id="toc" class="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> | |
| − | + | </li> | |
| − | + | <li class="toclevel-1 tocsetction-3"><a href="#3"><span class="tocnumber">3</span> <span class="toctext">Assembly | |
| − | + | 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-6"><a href="#4.1"><span class="tocnumber">4.1</span class="toctext">Protein expression and purification</span></a> | |
| − | + | </li> | |
| − | + | <li class="toclevel-2 tocsection-7"><a href="#4.2"><span class="tocnumber">4.2</span class="toctext">Electrophoretic Mobility shift assay</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> | |
| − | + | </li> | |
| − | + | </ul> | |
| − | + | </div> | |
| − | + | <section> | |
| − | + | <font size="5"><b>The PICasSO Toolbox </b> </font> | |
| − | + | <p><br></p> | |
| − | + | <div class="thumb"></div> | |
| − | + | <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" style="width:99%;" class="thumbimage"> | |
| − | + | <div class="thumbcaption"> | |
| − | + | <i><b>Figure 1: Example how the part collection can be used to engineer new staples</b></i> | |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| + | |||
| − | + | <p> | |
| − | + | <br> | |
| − | + | The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells, | |
| − | + | impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of | |
| − | + | chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely | |
| − | + | manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the | |
| − | + | 3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular | |
| − | + | toolbox based on various DNA-binding proteins to address this issue. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | The <b>PICasSO</b> part collection offers a comprehensive, modular platform for precise manipulation and | |
| − | + | re-programming | |
| − | + | of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic | |
| − | + | interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. | |
| − | + | Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and | |
| − | + | testing of new staples, ensuring functionality <i>in vitro</i> and <i>in vivo</i>. We took special care to include | |
| − | + | parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new 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 enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely | |
| − | + | new Cas staples in the future. We also include our simple staples that 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 the functionality of our Cas and Basic staples. These | |
| − | + | consist of | |
| − | + | protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling <i>in vivo</i>. | |
| − | + | Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our | |
| − | + | interkingdom conjugation system. <br> | |
| − | + | <b>(iii)</b> As the final component of our collection, we provide parts that support the use of 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 for functional | |
| − | + | readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | The following table gives a complete 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.<br> | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | <font size="4"><b>Our part collection includes:</b></font><br> | |
| − | + | </p> | |
| − | + | ||
| − | + | ||
| − | + | <table style="width: 90%;"> | |
| − | + | <td colspan="3" align="left"><b>DNA-binding proteins: </b> | |
| − | + | The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring | |
| − | + | easy assembly.</td> | |
| − | + | <tbody> | |
| − | + | <tr bgcolor="#FFD700"> | |
| − | + | <td><a href="https://parts.igem.org/Part:BBa_K5237000" target="_blank">BBa_K5237000</a></td> | |
| − | + | <td>fgRNA Entryvector MbCas12a-SpCas9</td> | |
| − | + | <td>Entryvector for simple fgRNA cloning via SapI</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <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 to form a functional staple, for example with fgRNA and dCas9 </td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <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 to form a functional staple, for example with our fgRNA or dCas12a | |
| − | + | </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 or fgRNA to bring two DNA strands in 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 taple: 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 colspan="3" align="left"><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>Cathepsin B which can be selectively express to cut the cleavable linker</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>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple | |
| − | + | units</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>Undergoes protein transsplicing after protease activation, can be used to create functionalized staple | |
| − | + | units</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td><a href="https://parts.igem.org/Part:BBa_K5237014" target="_blank">BBa_K5237014</a></td> | |
| − | + | <td>fgRNA processing casette</td> | |
| − | + | <td>Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing</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>Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large | |
| − | + | constructs</td> | |
| − | + | </tr> | |
| − | + | </tbody> | |
| − | + | <td colspan="3" align="left"><b>Readout Systems: </b> | |
| − | + | FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells | |
| − | + | enabling swift testing and easy development for new systems.</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>Donor part for the FRET assay binding the Oct1 binding cassette. 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. 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, can be used for different 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 Trans-Activator: NLS-Gal4-GFLG-VP64</td> | |
| − | + | <td>Readout system that responds to protease activity. It 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>NLS-Gal4-VP64</td> | |
| − | + | <td>Trans-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 Promotor, mCherry</td> | |
| − | + | <td>Readout system for enhancer binding. It 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>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. It was used as a luminescence readout for | |
| − | + | simulated enhancer hijacking.</td> | |
| − | + | </tr> | |
| − | + | </tbody> | |
| − | + | </table> | |
| − | + | </p> | |
| − | + | </section> | |
| − | + | <section id="1"> | |
| − | + | <h1>1. Sequence overview</h1> | |
| − | + | </section> | |
| − | + | </body> | |
| + | </html> | ||
| − | + | <!--################################--> | |
| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K5237007 SequenceAndFeatures</partinfo> | ||
| + | <!--################################--> | ||
| − | + | <html> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | |||
| − | + | <section id="2"> | |
| − | + | <h1>2. Usage and Biology</h1> | |
| − | + | <p> | |
| − | + | GCN4 is a yeast transcription factor from the bZip family of DNA-binding proteins, first discovered by McKnight and co-workers in 1988. | |
| − | + | The bZip motif features a coiled-coil leucine zipper dimerization domain paired with a highly charged basic region, which directly interacts | |
| − | + | with DNA. GCN4 binds specifically to the cyclic AMP response element (CRE) DNA sequence (5' ATGACGTCAT 3') in the promoter regions of target genes, primarily through its basic | |
| − | + | residues at the N-terminus. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | In our project, GCN4 was employed to study DNA-binding kinetics and develop a minimal "Mini staple" that brings two DNA target sites into proximity | |
| − | + | by binding them simultaneously. This "Mini staple" was designed as a versatile tool for precise DNA manipulation in synthetic biology applications. | |
| − | + | </p> | |
| − | + | <p> | |
| − | + | The DNA-binding properties of GCN4 were tested using an electrophoretic mobility shift assay (EMSA) to quantify binding affinity and kinetics. | |
| − | + | EMSA is a widely adopted method to study DNA-protein interactions. It works on the principle that nucleic acids bound to proteins exhibit reduced | |
| − | + | electrophoretic mobility compared to unbound nucleic acids (Hellman & Fried, 2007). EMSA can be employed both qualitatively, to assess DNA-binding | |
| − | + | capabilities, and quantitatively, to determine critical parameters such as binding stoichiometry and the apparent dissociation constant (K<sub>d</sub>) | |
| − | + | (Fried, 1989). | |
| − | + | </p> | |
| + | </section> | ||
| + | <section id="3"> | ||
| + | <h1>3. Assembly and part evolution</h1> | ||
| + | <p> | ||
| + | The GCN4 amino acid sequence was taken from literature (Hollenbeck et al. 2001) and codon optimized for <i>E. coli</i>. | ||
| + | A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an Enterokinase, if necessary. | ||
| + | The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using <i>E. coli</i> BL21 (DE3) cells. | ||
| + | </p> | ||
| + | </section> | ||
| + | <section id="4"> | ||
| + | <h1>4. Results</h1> | ||
| + | <section id="4.1"> | ||
| + | <h2>4.1 Protein expression and purification</h2> | ||
| + | <p>The FLAG-GCN4 protein could be readily expressed in <i>E. coli</i> BL21 (DE3). The protein was purified using an anti-FLAG resin. | ||
| + | Fractions taken during purification were analyzed by SDS-PAGE. Purified protein was quantified using a Lowry assay, 1.18 mg/mL were obtained, resulting in 153 µM of monomeric FLAG-GCN4. | ||
</p> | </p> | ||
| + | <div class="thumb"> | ||
| + | <div class="thumbinner" style="width:500px"> | ||
| + | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-sds-page-expression-validation.svg" style="width:99%;" class="thumbimage"> | ||
| + | <div class="thumbcaption"> | ||
| + | <i><b>Figure 2: SDS-PAGE analysis of FLAG-GCN4 purification</b> Fractions analysed are the raw lysate, flow through and eluate. | ||
| + | Depicted is GCN4 (this part), rGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237008" | ||
| + | target="_blank">BBa_K5237008</a>), and bGCN4 (<a href="https://parts.igem.org/Part:BBa_K5237009" | ||
| + | target="_blank">BBa_K5237009</a>)</i>. Protein size is indicated next to construct name and purified band with protein of interest highlighted by a red box. | ||
| + | </i> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
</section> | </section> | ||
| − | <section id=" | + | <section id="4.2"> |
| − | < | + | <h2>4.2 Electrophoretic Mobility shift assay</h2> |
<p> | <p> | ||
| − | The | + | The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein |
| − | + | interactions. EMSa functions on the basis that nucleic acids bound to proteins have reduced electrophoretic | |
| − | + | mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift | |
| + | assays can both be used to qualitatively assess DNA binding capabilities or quantitatively to determine binding | ||
| + | stoichiometry and kinetics such as the apparent dissociation constant (K<sub>d</sub>) (Fried, 1989). | ||
</p> | </p> | ||
| − | + | <div class="thumb"> | |
| − | + | <div class="thumbinner" style="width:40%;"> | |
| − | + | <img alt="" src="https://static.igem.wiki/teams/5237/figures-corrected/leucin-zipper-emsa.svg" | |
| − | + | style="width:99%;" class="thumbimage"> | |
| − | + | <div class="thumbcaption"> | |
| − | + | <i> | |
| − | + | <b>Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)</b> | |
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<p> | <p> | ||
| − | + | GCN4 was tested for its DNA-binding capabilities using an electrophoretic mobility shift assay (EMSA). The protein was incubated with a DNA probe containing the GCN4 binding site. The formation of a protein-DNA complex was analyzed by native PAGE. | |
| − | + | To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (<a href="https://parts.igem.org/Part:BBa:K5237008">BBa_K5237008</a>). | |
| − | + | 0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After | |
| + | electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The | ||
| + | obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex: | ||
| + | <br><br> | ||
| + | Θ<sub>app</sub> = Θ<sub>min</sub> + (Θ<sub>max</sub> - Θ<sub>min</sub>) × | ||
| + | (K<sub>a</sub><sup>2</sup> [L]<sub>tot</sub><sup>2</sup>) / (1 + K<sub>a</sub><sup>2</sup> | ||
| + | [L]<sub>tot</sub><sup>2</sup>) | ||
| + | <span style="float: right;">Equation 1</span> | ||
| + | <br><br> | ||
| + | Here [L]<sub>tot</sub> describes the total protein monomer concentration, K<sub>a</sub> | ||
| + | corresponds | ||
| + | to the apparent monomeric equilibration constant. The Θ<sub>min/max</sub> values are the | ||
| + | experimentally | ||
| + | determined site saturation values (For this experiment 0 and 1 were chosen for min and max | ||
| + | respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation | ||
| + | constant K<sub>k</sub> of (0.2930.033)×10<sup>-6</sup> M, which is almost identical to the | ||
| + | rGCN4 binding | ||
| + | affinity to INVii a <sub>d</sub> of (0.2980.030)×10<sup>-6</sup> M. | ||
</p> | </p> | ||
| + | <div class="thumb"> | ||
| + | <div class="thumbinner" style="width:500px"> | ||
| + | <img alt="" src="https://static.igem.wiki/teams/5237/wetlab-results/mist-leucine-zipper-kd-plot.svg" style="width:99%;" class="thumbimage"> | ||
| + | <div class="thumbcaption"> | ||
| + | <i><b>Figure 4: Quantitative EMSA</b>Quantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of | ||
| + | different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of | ||
| + | bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted | ||
| + | for each data point. Values are presented as mean +/- SD.</i> | ||
| + | </div> | ||
| + | </div> | ||
<p> | <p> | ||
| − | + | The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10<sup>-6</sup> M) and rGCN4 | |
| − | < | + | ((0.2980.030) × 10<sup>-6</sup> M) are |
| − | + | approximately a factor 10 higher then those described in literature ((96) × 10<sup>-8</sup> M for | |
| + | GCN4 and (2.90.8) × 10<sup>-8</sup> M for rGCN4) (Hollenbeck <i class="italic">et al.</i>, 2001). The | ||
| + | differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos. | ||
| + | Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity) bands in | ||
| + | the SDS-PAGE analysis, indicating the co-purification of small amounts of unspecific proteins. | ||
| + | <br><br> | ||
| + | The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due | ||
| + | to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity | ||
| + | between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds | ||
| + | C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the | ||
| + | dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the | ||
| + | FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed | ||
</p> | </p> | ||
| − | + | </section> | |
| − | + | <section id="5"> | |
| − | + | <h1>5. References</h1> | |
| − | + | <p>Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. <em>ELECTROPHORESIS, 10</em>(5–6), 366–376. <a href="https://doi.org/10.1002/elps.1150100515" target="_blank">https://doi.org/10.1002/elps.1150100515</a></p> | |
| − | + | ||
| − | + | <p>Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. <em>Nature Protocols, 2</em>(8), 1849–1861. <a href="https://doi.org/10.1038/nprot.2007.249" target="_blank">https://doi.org/10.1038/nprot.2007.249</a></p> | |
| − | + | <p>Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 Variant with a C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. <em>Biochemistry, 40</em>(46), 13833–13839.</p> | |
| − | + | ||
| − | + | <p>Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a Single Consensus Half-Site. <em>Biochemistry, 39</em>(21), 6380–6389. <a href="https://doi.org/10.1021/bi992705n" target="_blank">https://doi.org/10.1021/bi992705n</a></p> | |
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Revision as of 09:01, 30 September 2024
Staple subunit: GCN4
GCN4 is a yeast transcription factor belonging to the bZip family of DNA-binding proteins. We use GCN4 to study DNA-binding kinetics in our "Mini staples" that bring two DNA target sites into proximity by binding them simultaneously.
Contents
Figure 1: Example how the part collection can be used to engineer new staples
The 3D organization of the genome plays a crucial role in regulating gene expression in eukaryotic cells,
impacting cellular behavior, evolution, and disease. Beyond the linear DNA sequence, the spatial arrangement of
chromatin, influenced by DNA-DNA interactions, shapes pathways of gene regulation. However, the tools to precisely
manipulate this genomic architecture remain limited, rendering it challenging to explore the full potential of the
3D genome in synthetic biology. We - iGEM Team Heidelberg 2024 - have developed PICasSO, a powerful molecular
toolbox based on various DNA-binding proteins to address this issue.
The PICasSO part collection offers a comprehensive, modular platform for precise manipulation and re-programming of DNA-DNA interactions using protein staples in living cells, enabling researchers to recreate natural 3D genomic interactions, such as enhancer hijacking, or to design entirely new spatial architectures for gene regulation. Beyond its versatility, PICasSO includes robust assay systems to support the engineering, optimization, and testing of new staples, ensuring functionality in vitro and in vivo. We took special care to include parts crucial for testing every step of the cycle (design, build, test, learn) when engineering new parts
At its heart, the PICasSO part collection consists of three categories.
(i) Our DNA-binding proteins
include our
finalized enhancer hijacking Cas staple as well as half staples that can be used by scientists to compose entirely
new Cas staples in the future. We also include our simple staples that 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 the functionality of our Cas and Basic staples. These
consist of
protease-cleavable peptide linkers and inteins that allow condition-specific, dynamic stapling in vivo.
Besides staple functionality, we also include the parts to enable the efficient delivery of PICasSO's constructs with our
interkingdom conjugation system.
(iii) As the final component of our collection, we provide parts that support the use of 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 for functional
readout via a luciferase reporter, which allows for straightforward experimental simulation of enhancer hijacking.
The following table gives a complete 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.
Our part collection includes:
| DNA-binding proteins: The building blocks for engineering of custom staples for DNA-DNA interactions with a modular system ensuring easy assembly. | ||
| BBa_K5237000 | fgRNA Entryvector MbCas12a-SpCas9 | Entryvector for simple fgRNA cloning via SapI |
| BBa_K5237001 | Staple subunit: dMbCas12a-Nucleoplasmin NLS | Staple subunit that can be combined to form a functional staple, for example with fgRNA and dCas9 |
| BBa_K5237002 | Staple subunit: SV40 NLS-dSpCas9-SV40 NLS | Staple subunit that can be combined to form a functional staple, for example with our fgRNA or dCas12a |
| BBa_K5237003 | Cas-Staple: SV40 NLS-dMbCas12a-dSpCas9-Nucleoplasmin NLS | Functional Cas staple that can be combined with sgRNA or fgRNA to bring two DNA strands in 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 taple: 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 | Cathepsin B which can be selectively express to cut the cleavable linker |
| BBa_K5237012 | Caged NpuN Intein | Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units |
| BBa_K5237013 | Caged NpuC Intein | Undergoes protein transsplicing after protease activation, can be used to create functionalized staple units |
| BBa_K5237014 | fgRNA processing casette | Processing casette to produce multiple fgRNAs from one transcript, can be used for multiplexing |
| BBa_K5237015 | Intimin anti-EGFR Nanobody | Interkindom conjugation between bacteria and mammalian cells, as alternative delivery tool for large constructs | Readout Systems: FRET and enhancer recruitment to measure proximity of stapled DNA in bacterial and mammalian living cells enabling swift testing and easy development for new systems. |
| BBa_K5237016 | FRET-Donor: mNeonGreen-Oct1 | Donor part for the FRET assay binding the Oct1 binding cassette. 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. Can be used to visualize DNA-DNA proximity |
| BBa_K5237018 | Oct1 Binding Casette | DNA sequence containing 12 Oct1 binding motifs, can be used for different 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. It 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 Promotor, mCherry | Readout system for enhancer binding. It 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. It was used as a luminescence readout for simulated enhancer hijacking. |
1. Sequence overview
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
GCN4 is a yeast transcription factor from the bZip family of DNA-binding proteins, first discovered by McKnight and co-workers in 1988.
The bZip motif features a coiled-coil leucine zipper dimerization domain paired with a highly charged basic region, which directly interacts
with DNA. GCN4 binds specifically to the cyclic AMP response element (CRE) DNA sequence (5' ATGACGTCAT 3') in the promoter regions of target genes, primarily through its basic
residues at the N-terminus.
In our project, GCN4 was employed to study DNA-binding kinetics and develop a minimal "Mini staple" that brings two DNA target sites into proximity
by binding them simultaneously. This "Mini staple" was designed as a versatile tool for precise DNA manipulation in synthetic biology applications.
The DNA-binding properties of GCN4 were tested using an electrophoretic mobility shift assay (EMSA) to quantify binding affinity and kinetics.
EMSA is a widely adopted method to study DNA-protein interactions. It works on the principle that nucleic acids bound to proteins exhibit reduced
electrophoretic mobility compared to unbound nucleic acids (Hellman & Fried, 2007). EMSA can be employed both qualitatively, to assess DNA-binding
capabilities, and quantitatively, to determine critical parameters such as binding stoichiometry and the apparent dissociation constant (Kd)
(Fried, 1989).
The GCN4 amino acid sequence was taken from literature (Hollenbeck et al. 2001) and codon optimized for E. coli.
A FLAG-tag (DYKDDDDK) was added to the N-terminus for protein purification. The FLAG-tag can be cleaved off using an Enterokinase, if necessary.
The FLAG-GCN4 sequence was cloned into a T7 expression vector and expressed using E. coli BL21 (DE3) cells.
The FLAG-GCN4 protein could be readily expressed in E. coli BL21 (DE3). The protein was purified using an anti-FLAG resin.
Fractions taken during purification were analyzed by SDS-PAGE. Purified protein was quantified using a Lowry assay, 1.18 mg/mL were obtained, resulting in 153 µM of monomeric FLAG-GCN4.
The Electrophoretic mobility shift assay (EMSA) is a widely adopted method used to study DNA-protein
interactions. EMSa functions on the basis that nucleic acids bound to proteins have reduced electrophoretic
mobility, compared to their counterpart. (Hellman & Fried, 2007). Mobility-shift
assays can both be used to qualitatively assess DNA binding capabilities or quantitatively to determine binding
stoichiometry and kinetics such as the apparent dissociation constant (Kd) (Fried, 1989).
GCN4 was tested for its DNA-binding capabilities using an electrophoretic mobility shift assay (EMSA). The protein was incubated with a DNA probe containing the GCN4 binding site. The formation of a protein-DNA complex was analyzed by native PAGE.
To further analyze DNA binding, quantitative shift assays were performed for GCN4 and rGCN4 (BBa_K5237008).
0.5 µM DNA were incubated with varying concentrations of protein until equilibration. After
electrophoresis, bands were stained with SYBR-Safe and quantified based on pixel intensity. The
obtained values were fitted to equation 1, describing formation of a 2:1 protein-DNA complex:
The apparent binding kinetics calculated for GCN4 ((0.2930.033) × 10-6 M) and rGCN4
((0.2980.030) × 10-6 M) are
approximately a factor 10 higher then those described in literature ((96) × 10-8 M for
GCN4 and (2.90.8) × 10-8 M for rGCN4) (Hollenbeck et al., 2001). The
differences could be due to the lower sensitivity of SYBR-Safe staining compared to radio-labeled oligos.
Most likely, the protein concentration was miscalculated due to the presence of additional (lower intensity) bands in
the SDS-PAGE analysis, indicating the co-purification of small amounts of unspecific proteins.
Fried, M. G. (1989). Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. ELECTROPHORESIS, 10(5–6), 366–376. https://doi.org/10.1002/elps.1150100515 Hellman, L. M., & Fried, M. G. (2007). Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nature Protocols, 2(8), 1849–1861. https://doi.org/10.1038/nprot.2007.249 Hollenbeck, J. J., Gurnon, D. G., Fazio, G. C., Carlson, J. J., & Oakley, M. G. (2001). A GCN4 Variant with a C-Terminal Basic Region Binds to DNA with Wild-Type Affinity. Biochemistry, 40(46), 13833–13839. Hollenbeck, J. J., & Oakley, M. G. (2000). GCN4 Binds with High Affinity to DNA Sequences Containing a Single Consensus Half-Site. Biochemistry, 39(21), 6380–6389. https://doi.org/10.1021/bi992705n2. Usage and Biology
3. Assembly and part evolution
4. Results
4.1 Protein expression and purification
Figure 2: SDS-PAGE analysis of FLAG-GCN4 purification Fractions analysed are the raw lysate, flow through and eluate.
Depicted is GCN4 (this part), rGCN4 (BBa_K5237008), and bGCN4 (BBa_K5237009). Protein size is indicated next to construct name and purified band with protein of interest highlighted by a red box.
4.2 Electrophoretic Mobility shift assay
Figure 3: Overview Image of Electrophoretic Mobility Shift Assay (EMSA)
Θapp = Θmin + (Θmax - Θmin) ×
(Ka2 [L]tot2) / (1 + Ka2
[L]tot2)
Equation 1
Here [L]tot describes the total protein monomer concentration, Ka
corresponds
to the apparent monomeric equilibration constant. The Θmin/max values are the
experimentally
determined site saturation values (For this experiment 0 and 1 were chosen for min and max
respectively). GCN4 binds to its optimal DNA binding motif with an apparent dissociation
constant Kk of (0.2930.033)×10-6 M, which is almost identical to the
rGCN4 binding
affinity to INVii a d of (0.2980.030)×10-6 M.
Figure 4: Quantitative EMSAQuantitative assessment of binding affinity for GCN4 and rGCN4. Proteins of
different concentrations were incubated with 0.5 µM DNA until equilibrium and fraction bound analyzed, after gel electrophoresis, by dividing pixel intensity of
bound fraction with pixel intensity of bound and unbound fraction. At least three separate measurements were conducted
for each data point. Values are presented as mean +/- SD.
The FLAG-tag fusion to the N-terminus of proteins could potentially decrease binding affinity, likely due
to steric hindrance affecting the interaction with DNA. Interestingly, the differences in binding affinity
between GCN4 and rGCN4 appear negligible. Since GCN4 binds to DNA via its N-terminus and rGCN4 binds
C-terminally, the FLAG-tag likely does not directly influence DNA binding. However, it may influence the
dimerization of the proteins, which is necessary for DNA binding. To further investigate this, the
FLAG-tag can be cleaved using an enterokinase and potential changes in binding affinity analyzed
5. References