Team:UiOslo Norway/Experiments

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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).

Protocols

Molecular biology

• PCR with Phusion-HF DNA Polymerase (NEB)

• PCR with Taq DNA Polymerase (NEB)

• Restriction Digest

• Shrimp Alkaline Phosphatase Treatment (Fermentas)

• Ligation with T4 DNA Polymerase (NEB)

• TOPO Zero blunt cloning (Invitrogen)

• Wizard® SV Gel and PCR Clean-Up System (Promega)

• QIAprep Spin Miniprep Kit (Qiagen)

• NucleoSpin® Tissue (Machery-Nagle)

• In vitro Mutagenesis

• Agarose Gel

• List of primers

Protein Biochemistry

• Protein Expression

• Solubility Assay

• SDS-Page

Handling bacteria

• E. coli strains

• Transformation of chemically competent E. coli cells

• Antibiotic Concentrations

• LB media

• Cultivation of Methylococcus capsulatus (Bath)

• NMS medium

• NMS-medium for growth of Methylococcus capsulatus (Bath)