<h4>Golden Gate assembly with CAM insert and pSEVA backbone</h4>
<h4>Golden Gate Assembly with Complementation systems and Ribo J</h4>
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<h5>Purpose:</h5>
<p>To create our pSEVA3411 plasmid, which will be the backbone for the final plasmid coding our system</p>
<h5>Purpose:</h5>
<p>To create our “B” cassette, which functioned as the complementing portion of our complex.</p>
<h5>Procedure:</h5>
<ol>
<li>We performed golden gate assembly using BsaI-HFv2 and T4 ligase to combine our CAM insert and our pSEVA backbone.</li>
<li>Remember that these steps need to be detailed enough that someone can reproduce our work</li>
<li>TBD</li>
</ol>
<h5>Results:</h5>
<ul>
<li>Final concentration: 23.15 ng/µL</li>
<li>pSEVA3411 plasmid backbone is available on the iGEM parts registry as <ahref = "http://parts.igem.org/Part:BBa_K4215000">BBa_K4215000</a></li>
<li>After running the products on a gel, the lengths did not correspond to the expected length of a fully assembled complex. At best, we only got partial assembly, of just the RiboJ and one half of the complementation system. </li>
</ul>
<h5>Pictures:</h5>
<imgsrc="https://static.igem.wiki/teams/4215/wiki/b-ladder.jpg"class="img-fluid"alt="HRP, 100kb ladder, Rluciferase, Beta lactamase, TEV protease(right to left)">
<p>HRP, 100kb ladder, Rluciferase, Beta lactamase, TEV protease(right to left). Highest bands all represent lengths that correspond to one fragment of the complementation system. This gel helped us to recognize that an error was made in the initial design of the riboJ complex in which the 5’ BbsI cut site left an incompatible overhang, which prevented the assembly from joining with the N-terminal split proteins. </p>
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<buttonclass="btn btn-primary btn-lg btn-block collapsed"data-bs-toggle="collapse"data-bs-target="#collapseExpOne"aria-expanded="false"aria-controls="collapseExpOne">Golden Gate Assembly with pSEVA3411 and D-promoter</button>
<p>During the course of our experimental wet lab phase, we designed a genetic construct to be assembled and produced in <i>E. coli.</i> To create this, we utilized Combinatorial Golden Gate assembly to produce four unique genetic cassettes, each encoding a specific portion of our biosensor complex. Each cassette existed as a plasmid, with Cassettes A and C each encoding one half of the binder/linker portion of our construct. Cassette B encoded our split complementation system, which “sandwiched” our RiboJ sequence. Cassette D encoded the backbone of our final plasmid that would be transfected into cells and translated into protein. This cassette included our selected resistances as well as a D-Promoter.</p>
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<h2>Future Steps</h2>
<p></p>
<p>After initial experimentation, we discovered that our RiboJ sequence was not functioning correctly in the Golden Gate assembly, so we needed to fix one end of the riboJ sequence and rerun the Golden Gate assembly. Furthermore, a wise step would be to increase the number of split reporter proteins we are experimenting with, as we gathered many more during the research phase that we never got to test. This pursuit would also adjust split sites and substrate concentrations to maximize signaling.</p>
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<h1>Computational Summary</h1>
<p>There are many biomarkers associated with concussions and mTBI, but their binding interactions with particular proteins are not often studied so some forms of binding (particularly the strength of the biomarker/binder complex) are difficult to characterize using only the literature. After narrowing the field of likely biomarkers and binders from our literature search, we obtained sequences and then used AlphaFold<sup><ahref = "#Ref1">1</a></sup> to model complexes between binders and biomarkers. Based on the results from alphafold, we also determined binding effectiveness with two different docking suites, HDOCK and ZDOCK. Based on the results of all these simulations, we decided to use UCHL1 as our biomarker and split ubiquitin as our binder. GFAP had some promising binders as well, but we ultimately decided to focus purely on UCHL1 since we were unable to express GFAP.</p>
