Part:BBa_K3888011
[Ni-Fe] Hydrogenase Core gene HoxG1 and HoxK1
This composite part is used for heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli.
Hydrogenase is extremely sensitive to oxygen. By expressing oxygen-tolerant hydrogenase in E.coli, the oxygen tolerance will increase thus increases the amount of hydrogen produced by hydrogenase in E.coli. The strain of E.coli we used in the experiments is E.coli BL21. The part basically consists of two main parts: HoxG1(BBa_K3888003) and HoxK1(BBa_K3888004), which encode the large subunit and small subunit of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase respectively.
Other key features also include a natural (wild) type of RBS, B0032m, and a pTac promoter which is controlled by LacI(induced by IPTG) and lac operator .
Figure 1: The Oxygen-tolerant Hydrogenase introduction plasmid constructed, which has a length of 8261bp.
Applications of BBa_K3888011
According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows:
Figure 2. Hydrogenase plasmid. Left: Plasmid design. Right: Plasmid Agarose Gel (0.8%)
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:
Figure 3. The 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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 363
Illegal XbaI site found at 465
Illegal SpeI site found at 531 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 531
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 678
Illegal BamHI site found at 1593
Illegal BamHI site found at 2932
Illegal XhoI site found at 1915 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 363
Illegal XbaI site found at 465
Illegal SpeI site found at 531 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 363
Illegal XbaI site found at 465
Illegal SpeI site found at 531
Illegal AgeI site found at 1809 - 1000COMPATIBLE WITH RFC[1000]
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