Update file experiments.html

wiki/pages/experiments.html

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           <div class="h1">Plasmid Design and Construction</div>
-        <p>Plasmids were designed to express fluorescent proteins fused to various targeting sequences in <i>Saccharomyces cerevisiae</i> and <i>Chlamydomonas reinhardtii</i>. These sequences included uTP1/2 (UCYN-A transit peptide, identified through bioinformatics analysis), MTS1 (mitochondrial targeting sequence 1) <a href="#cite13">[13]</a>, and CTS (chloroplast targeting sequence) <a href="#cite14">[14]</a>. The uTP1/2 sequence was inserted at the C-terminal end of the fluorescent proteins, while MTS1 and CTS were incorporated at the N-terminal end. For uTP1/2 constructs, a His6-tag was incorporated between the transit peptide and the fluorescent protein to facilitate future purification experiments.</p>
+        <p>Plasmids were designed to express fluorescent proteins fused to various targeting sequences in <i>Saccharomyces cerevisiae</i> and <i>Chlamydomonas reinhardtii</i>. These sequences included uTP1/2 (UCYN-A transit peptide, identified through bioinformatics analysis), MTS1 (mitochondrial targeting sequence 1) <a href="#cite13">[13]</a>, mTP (mitochondrial transit peptide)<a href="#cite38">[38]</a> and cTP (chloroplast transit peptide) <a href="#cite14">[14]</a>. The uTP1/2 sequence was inserted at the C-terminal end of the fluorescent proteins, while MTS1, mTP and cTP were incorporated at the N-terminal end. For uTP1/2 constructs, a His6-tag was incorporated between the transit peptide and the fluorescent protein to facilitate future purification experiments.</p>
         <p>Two existing vectors with fluorescent proteins were used as backbones for this study. For <i>S. cerevisiae</i>, we utilized pUDE1311 <a href="#cite11">[11]</a>, which contains mNeonGreen and a URA3 marker for selection (full genotype: ConLS-ScHHF2p-ymNeonGreen-ScENO1t-ConR1-URA3-2µ-AmpR). This vector was kindly provided by Marcel Vieira Lara of TU Delft. For <i>C. reinhardtii</i>, we used pOpt2 <a href="#cite9">[9]</a> <a href="#cite10">[10]</a>, containing mVenus and a ble marker for selection. This plasmid, kindly provided by Friedrich Kleiner from the MEA lab at TU Delft, also features specific introns in the mVenus coding sequence to prevent gene silencing. Both plasmids contain an ampicillin resistance gene for selection in <i>E. coli</i>. The complete sequences of both unmodified vectors and plasmids with all inserts have been made publicly available [link].</p>
-        <p>All plasmids, inserts, and primers were designed in SnapGene (www.snapgene.com). The inserts containing the transit peptides were synthesized by GenScript (Piscataway, NJ, USA). Gibson Assembly was employed for plasmid construction. Primers with specific overhangs were designed for PCR amplification of inserts and linearization of plasmid backbones at both the C-terminal and N-terminal regions, for inserting uTP1/2 and MTS1/CTS respectively.</p>
-        <p>PCR amplification was performed using KOD polymerase. To eliminate leftover circular plasmid from the backbone amplification process, DpnI digestion was utilized. Gibson Assembly was then carried out to combine the linearized plasmid backbones with the uTP, MTS1, and CTS inserts using the Gibson Assembly® Cloning Kit by New England Biolabs <a href="#cite1">[1]</a>, following the manufacturer’s protocol <a href="#cite2">[2]</a>. pUC19 and a DNA fragment provided by the kit were used as positive controls.</p>
+        <p>All plasmids, inserts, and primers were designed in SnapGene (www.snapgene.com). The inserts containing the transit peptides were synthesized by GenScript (Piscataway, NJ, USA). Gibson Assembly was employed for plasmid construction. Primers with specific overhangs were designed for PCR amplification of inserts and linearization of plasmid backbones at both the C-terminal and N-terminal regions, for inserting uTP1/2 and MTS1/mTP/cTP respectively.</p>
+        <p>PCR amplification was performed using KOD polymerase. To eliminate leftover circular plasmid from the backbone amplification process, DpnI digestion was utilized. Gibson Assembly was then carried out to combine the linearized plasmid backbones with the uTP, MTS1, mTP and cTP inserts using the Gibson Assembly® Cloning Kit by New England Biolabs <a href="#cite1">[1]</a>, following the manufacturer’s protocol <a href="#cite2">[2]</a>. pUC19 and a DNA fragment provided by the kit were used as positive controls.</p>
 
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             <li id="cite35">Cabau, C., Escudié, F., Djari, A., Guiguen, Y., Bobe, J., & Klopp, C. (2017). Compacting and correcting Trinity and Oases RNA-Seq de novo assemblies. PeerJ, 5, e2988. <a href="https://doi.org/10.7717/peerj.2988" style="color:#185A4F;">https://doi.org/10.7717/peerj.2988</a></li>
             <li id="cite36">Emms, D. M., & Kelly, S. (2019). OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biology, 20(1). <a href="https://doi.org/10.1186/s13059-019-1832-y" style="color:#185A4F;">https://doi.org/10.1186/s13059-019-1832-y</a></li>
             <li id="cite37">iGEM Kaiserslautern & iGEM Sorbonne Universite. (2023). THE CHLAMY GUIDE iGEM Kaiserslautern & iGEM Sorbonne Universite. <a href="https://static.igem.org/mediawiki/2019/8/89/T--Sorbonne_U_Paris--TheChlamyGuide.pdf" style="color:#185A4F;">https://static.igem.org/mediawiki/2019/8/89/T--Sorbonne_U_Paris--TheChlamyGuide.pdf</a></li>
+            <li id="cite38"> <a href="https://parts.igem.org/Part:BBa_K4806011" style="color:#185A4F">https://parts.igem.org/Part:BBa_K4806011</a></li>
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