Difference between revisions of "Team:UiOslo Norway/Experiments"
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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> | 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> | ||
| − | 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. | + | 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> |
| + | 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>. | ||
Revision as of 10:50, 1 September 2015
Experiments
1. Obtaining the genes
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. For the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) we got the genes for the subunits (mmoXYZBCD) cloned into the BioBrick standard vector pSC1B3 from the iGEM 2014 team from Braunschweig, Germany. Those six parts are registered as BioBrick parts under the names; Bba_K1390001 (mmoB), Bba_K1390002 (mmoC), Bba_K1390003 (mmoD), Bba_K1390004 (mmoX), Bba_K1390005 (mmoY), and Bba_K1390006 (mmoZ). Genes encoding the enzymes for the conversion of methanol into biomass (medh2, hps, and phi) were amplified by PCR from Bacillus methanolicus (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. TOPO blunt end cloning of mmoG did not succeed. Instead mmoG was synthesized by IDT as a gBlock gene fragment, codon optimized for protein expression in E. coli.