Coding

Part:BBa_K3888003:Experience

Designed by: Linhan Lei   Group: iGEM21_SJTang   (2021-10-01)
Revision as of 11:26, 20 October 2021 by RyanLei (Talk | contribs) (→‎Applications of BBa_K3888003)


Applications of BBa_K3888003

According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows: Experiments hydrogenase.png

Both HoxG1 (Hydrogenase Large subunit) and HoxK1 (Hydrogenase small subunit) are from Hydrogenovibrio marinus MH-110. The signal sequence of HoxK1 is replaced with HyaA (Hydrogenase small subunit) signal sequence from E. coli BL21. The agarose gel shows the length of the plasmid is right. Then we transformed the obtained plasmid into E. coli BL21. Cultivate the seed solution overnight, and then inoculate it into 100 mL LB liquid medium at a ratio of 1:50. Two groups start induction at 0h with 1mM IPTG. After culturing for 8 hours at 37 °C 220rpm, 1 mM IPTG is added for overnight induction at 18 °C. Afterwards, the bacteria were collected and disrupted. Since Hydrogenase is a membrane protein, the cell disruption suspension and the centrifuged supernatant were collected separately for protein SDS-PAGE electrophoresis. The results of electrophoresis are as follows: Electrophoresis hydrogenase.png

Figure 1. Hydrogenase plasmid. Left: Plasmid design. Right: Plasmid Agarose Gel (0.8%)

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