Difference between revisions of "Team:UiOslo Norway/Experiments"
| Line 37: | Line 37: | ||
</br> | </br> | ||
</br> | </br> | ||
| − | Figure about construct: T7 prom—RBS—GENE Coming soon | + | <b>Figure about construct: T7 prom—RBS—GENE Coming soon</b> |
</br> | </br> | ||
</br> | </br> | ||
| Line 123: | Line 123: | ||
</tr> | </tr> | ||
</p></table> | </p></table> | ||
| + | |||
| + | <h2>4. Protein Expression and Solubility</h2> | ||
| + | |||
| + | <p>After generating expression constructs for each gene individually we tested if they could be expressed successful in <i>E. coli</i>. Furthermore it is necessary to show if the heterologous expressed proteins are soluble expressed and not aggregated into inclusion bodies. Aggregation in inclusion bodies prevents a proper folding and thereby proper function of the protein. To test the solubility of heterologous expressed proteins, proteinexpression is performed for a defined period of time. Afterwards the cells are harvested and disrupted (e.g. sonication, lysozyme, glass beads). By centrifugation the lysate is separated into an insoluble fraction, which contains cell material and inclusion bodies, and a soluble fraction, containing the cytoplasmic fraction including most proteins. Both fractions can be analysed via SDS-Page. | ||
| + | </p> | ||
| + | |||
| + | <h2>5. Characterization of NAD+ dependent methanol dehydrogenase (medh2)</h2> | ||
| + | |||
| + | <p> | ||
| + | |||
| + | </p> | ||
| + | |||
</div> | </div> | ||
Revision as of 12:10, 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.
2. Construction of BioBrick parts
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 mmoG as well as all three genes encoding the enzymes for the methanol to biomass conversion (medh2, hps, and phi) were created as BioBrick parts. The CDS of the hps 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 in vitro mutagenesis both restriction sites were removed and hps was cloned into pSC1B3 and submitted as a BioBrick part.
3. Generation of expression constructs
Before assembling the final constructs we wanted to show that each individual protein could be expressed in E. coli. 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. Figure about construct: T7 prom—RBS—GENE Coming soon 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).
Table 1: Details about the expression constructs
| Gene | 5' Restriction Site | 3' Restriction Site | Vector | Protein Tag |
|---|---|---|---|---|
| mmoX | NdeI | EcoRI | pET-30 | -- |
| mmoY | NdeI | EcoRI | pET-30 | -- |
| mmoZ | NdeI | EcoRI | pET-30 | -- |
| mmoB | NdeI | EcoRI | pET-30 | -- |
| mmoC | NdeI | EcoRI | pET-30 | -- |
| mmoD | NdeI | EcoRI | pET-30 | -- |
| mmoG | NdeI | EcoRI | pET-30 | -- |
| medh2 | EcoRI | XhoI | pET-28 | 6xHis-tag N-terminal |
| hps | NcoI | EcoRI | pET-30 | -- |
| phi | NdeI | EcoRI | pET-30 | -- |
4. Protein Expression and Solubility
After generating expression constructs for each gene individually we tested if they could be expressed successful in E. coli. Furthermore it is necessary to show if the heterologous expressed proteins are soluble expressed and not aggregated into inclusion bodies. Aggregation in inclusion bodies prevents a proper folding and thereby proper function of the protein. To test the solubility of heterologous expressed proteins, proteinexpression is performed for a defined period of time. Afterwards the cells are harvested and disrupted (e.g. sonication, lysozyme, glass beads). By centrifugation the lysate is separated into an insoluble fraction, which contains cell material and inclusion bodies, and a soluble fraction, containing the cytoplasmic fraction including most proteins. Both fractions can be analysed via SDS-Page.
5. Characterization of NAD+ dependent methanol dehydrogenase (medh2)