Team:SJTang/Engineering
Engineering Success
Cyanobacteria are prokaryotic organisms that can be used for photosynthesis, depending on their characteristics of hydrogen produced by nitrogen fixation, and in order to further improve the hydrogen production efficiency of E. coli, we chose a membrane-binding hydrogenation enzyme Oxygen-tolerant [NiFe]-hydrogenase from Hydrogenovibrio marinus MH-110. The structure of this hydrogenase is similar to that in Escherichia coli, and it is also composed of two subunits. Among them, HoxG1(NCBI Locus: WP_029908155) is the large subunit of hydrogenase, and HoxK1 (NCBI Locus: WP_029908158) is the small subunit. The structure information of the two of them on NCBI is as follows:
Figure.1 Conserved domain of Hydrogenase. Up: HoxG1. Down: HoxK1
Furthermore, in the process of reviewing papers, we found that the first 132 bp of the HyaA and HoxK1 sequences both contain the signal sequence. In order for this protein to be successfully expressed heterologously in E. coli, we replaced the signal sequence of HoxK1 and replaced it with the signal sequence of HyaA. Finally, we have the following plasmid design:
Figure.2Plasmid design of Hydrogenase production plasmid.
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:
Figure.3The SDS-PAGE result of cell disruption.
The results of electrophoresis showed that the expression of hydrogenase was successful and basically consistent with expectations. The results showed that the induction effect was better from 0h than 8h, and the content of HoxK1 and HoxG1 in the suspension was higher than that in the supernatant after centrifuge.