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| − | <h1> Experiments </h1>
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| − | <h2> 1. Obtaining the genes </h2>
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| − | <p>The first project part pursues the goal to get or clone all genes that are involved in our project into a plasmid, which allows a rapid and easy amplification of those genes for further experiments. </br>
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| − | For the soluble methane monooxygenase (sMMO) from <i>Methylococcus capsulatus</i> (Bath) we got the genes for the subunits (<i>mmoXYZBCD</i>) cloned into the BioBrick standard vector pSC1B3 from the
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| − | <a href="https://2014.igem.org/Team:Braunschweig/Achievements-content#parts" >
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| − | iGEM 2014 team from Braunschweig, Germany.
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| − | </a>
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| − | Those six parts are registered as BioBrick parts under the names; Bba_K1390001 (<i>mmoB</i>), Bba_K1390002 (<i>mmoC</i>), Bba_K1390003 (<i>mmoD</i>), Bba_K1390004 (<i>mmoX</i>), Bba_K1390005 (<i>mmoY</i>), and Bba_K1390006 (<i>mmoZ</i>). </br>
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| − | Genes encoding the enzymes for the conversion of methanol into biomass (<i>medh2</i>, <i>hps</i>, and <i>phi</i>) were amplified by PCR from <i>Bacillus methanolicus</i> (MGA3) genomic DNA and TOPO blunt end cloning into the pCR4 vector was performed. Primers were designed in a way that they bind in the 5’ and 3’ untranslated region (UTR) of each gene.</br>
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| − | TOPO blunt end cloning of <i>mmoG</i> did not succeed. Instead <i>mmoG</i> was synthesized by IDT as a gBlock gene fragment, codon optimized for protein expression in <i>E. coli</i>.
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| − | </p>
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| − | <h2> 2. Construction of BioBrick parts</h2>
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| − | <p>The second project part had the intention to create four new basic BioBrick parts. Those basic parts consist of the coding sequences (CDS) of a gene. The codonoptimized <i>mmoG</i> as well as all three genes encoding the enzymes for the methanol to biomass conversion (<i>medh2</i>, <i>hps</i>, and <i>phi</i>) were created as
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| − | <a href="https://2015.igem.org/Team:UiOslo_Norway/Basic_Part" >
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| − | BioBrick parts.
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| − | </a> </br>
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| − | The CDS of the <i>hps</i> gene, encoding the 3-hexulose-6-phosphate synthase, contains PstI and XbaI restriction sites making it not compatible with the BioBrick system. In two rounds of <i>in vitro</i> mutagenesis both restriction sites were removed and <i>hps</i> was cloned into pSC1B3 and submitted as a
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| − | <a href="https://2015.igem.org/Team:UiOslo_Norway/Basic_Part" >
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| − | BioBrick part.
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| − | </a>
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| − | </p>
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| − | <h2>3. Generation of expression constructs</h2>
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| − | <p>Before assembling the final constructs we wanted to show that each individual protein could be expressed in <i>E. coli</i>. The pET system has been chosen as our preferred system for overexpression of each individual protein. The vector backbones pET-28 and pET-30 were chosen as potential expression vector.</br>
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| − | </br>
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| − | </br>
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| − | Figure about construct: T7 prom—RBS—GENE Coming soon
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| − | </br>
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| − | </br>
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| − | With PCR we added restriction enzyme sites at the 5’ and 3’ end of CDS of each gene. Afterwards the gene was cloned either into pET-30 or pET-28 with the use of the listed restriction enzymes (Table 1 coming soon).
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| − | </p>
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| − | </div>
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| − | <div style="float:left; width:50%;">
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| − | <h1> Protocols </h1>
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| − | <h2> Molecular biology </h2>
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| − | <ul>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/PCR_phusion">PCR with Phusion-HF DNA Polymerase (NEB) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/PCR_taq">PCR with Taq DNA Polymerase (NEB) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Restriction_Digest">Restriction Digest </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Alkaline_Phospatase">Shrimp Alkaline Phosphatase Treatment (Fermentas) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Ligation">Ligation with T4 DNA Polymerase (NEB) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/TOPO_cloning">TOPO Zero blunt cloning (Invitrogen) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/PCR_Cleanup">Wizard® SV Gel and PCR Clean-Up System (Promega) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Miniprep">QIAprep Spin Miniprep Kit (Qiagen) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/NucleoSpin_Tissue">NucleoSpin® Tissue (Machery-Nagle) </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Mutagenesis">In vitro Mutagenesis </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Agarose_Gel">Agarose Gel </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Primers">List of primers </a></p></li>
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| − | </ul>
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| − | <h2> Protein Biochemistry </h2>
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| − | <ul>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Protein_Expression">Protein Expression </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Solubility_Assay">Solubility Assay </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/SDS-Page">SDS-Page </a></p></li>
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| − | </ul>
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| − | <h2>Handling bacteria </h2>
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| − | <ul>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/E_coli_strains"><i>E. coli</i> strains </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Transformation">Transformation of chemically competent <i>E. coli</i> cells</a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Antibiotics">Antibiotic Concentrations</a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/LB-media">LB media </a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/Cultivation_M_c(bath)">Cultivation of <i>Methylococcus capsulatus</i> (Bath)</a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/NMS medium">NMS medium</a></p></li>
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| − | <li><p><a href="https://2015.igem.org/Team:UiOslo_Norway/Experiments/NMS-medium_M_c(bath)">NMS-medium for growth of <i>Methylococcus capsulatus</i> (Bath)</a></p></li>
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| − | </ul>
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