<h2id="evolution"style="text-align: center; margin-top: 60px;">2. The <spanclass="vbg">Evolution.T7</span> system 🧬</h2>
<hr>
<p>In 2021, the <ahref="https://2021.igem.org/Team:Evry_Paris-Saclay">iGEM Evry Paris-Saclay</a> team developed the Evolution.T7 project. As four of our team members also participated in the 2021 team: two students, Doriane Blaise and Georges Sainte-Rose, and two PIs, Ioana Popescu and Manish Kushwaha, we decided to use this powerful system for <i>in vivo</i> evolution of the selected microbial opsins.</p>
<p>In 2021, the <ahref="https://2021.igem.org/Team:Evry_Paris-Saclay"target="_blank"rel="noopener noreferrer">iGEM Evry Paris-Saclay</a> team developed the Evolution.T7 project. As four of our team members also participated in the 2021 team: two students, Doriane Blaise and Georges Sainte-Rose, and two PIs, Ioana Popescu and Manish Kushwaha, we decided to use this powerful system for <i>in vivo</i> evolution of the selected microbial opsins.</p>
<p>Evolution.T7 is an <i>in vivo</i> directed evolution system that uses the orthogonal T7 RNA polymerase (T7RNAP) fused at the N-terminus with different cytosine or adenine deaminases (CD, AD), base deaminases (BD) for simplicity. T7RNAP is highly specific to its promoter sequence (TAATACGACTCACTATA), the strength of which is increased if it is followed by GGG at 3' end <b>[9]</b>. The T7RNAP initiates the transcription at the first guanidine of this stretch of three G and terminates it at specific terminator(s) <b>[10]</b>. When BD-T7RNAP fusion protein is expressed in <i>E. coli</i>, the sequence flanked by the T7 promoter and the T7 terminator(s) gets mutated as the BD deaminates randomly the nucleotides on the non template strand of the T7RNAP <b>[11]</b>. In an <i>E. coli</i> strain deficient in its natural DNA repairing system (Δ<i>ung</i> Δ<i>nfi</i>), upon DNA replication, these deaminated bases lead to C->T or A->G transition mutations, depending on whether CD or AD was used.</p>
...
...
@@ -401,7 +401,7 @@
<ul>
<li>The HR is covalently linked to the NpHtrII transmembrane domain (via a short linker sequence) to ensure their colocalization</li>
<li>The natural histidine kinase domain of the NpHtrII was removed as it does not specifically interact with an <i>E. coli</i> signaling cascade. It was replaced by an endogenous <i>E. coli</i> histidine kinase domain.</li>
<li>The <i>E. coli</i> EnvZ was chosen as a histidine kinase domain as it is part of the widely known EnvZ-OmpR two component system <b>[21]</b> (described above on this page in chapter n°3, but also in the <ahref="https://2023.igem.wiki/evry-paris-saclay/contribution">Contribution</a> page on this wiki).</li>
<li>The <i>E. coli</i> EnvZ was chosen as a histidine kinase domain as it is part of the widely known EnvZ-OmpR two component system <b>[21]</b> (described above on this page in chapter n°3, but also in the <ahref="https://2023.igem.wiki/evry-paris-saclay/contribution"target="_blank"rel="noopener noreferrer">Contribution</a> page on this wiki).</li>
