Difference between revisions of "Part:BBa K3888003:Experience"
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According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows: | According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows: | ||
[[File:Experiments hydrogenase.png]]<br> | [[File:Experiments hydrogenase.png]]<br> | ||
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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. | 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: | 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: | ||
| − | [[File:Electrophoresis hydrogenase]] | + | [[File:Electrophoresis hydrogenase.png]] |
===Figure 1. Hydrogenase plasmid. Left: Plasmid design. Right: Plasmid Agarose Gel (0.8%)=== | ===Figure 1. Hydrogenase plasmid. Left: Plasmid design. Right: Plasmid Agarose Gel (0.8%)=== | ||
Revision as of 11:26, 20 October 2021
Applications of BBa_K3888003
According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows:
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 1. Hydrogenase plasmid. Left: Plasmid design. Right: Plasmid Agarose Gel (0.8%)
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