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> | ||
| − | ===Figure 1. 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. | + | ===Figure 1. 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.<br> | 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.<br> | ||
The results of electrophoresis are as follows: | The results of electrophoresis are as follows: | ||
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===User Reviews=== | ===User Reviews=== | ||
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| − | + | 1. Kim, Jaoon YH, Byung Hoon Jo, and Hyung Joon Cha. "Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli." Journal of biotechnology 155.3 (2011): 312-319. | |
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Latest revision as of 19:50, 20 October 2021
Applications of BBa_K3888003
According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows:
Figure 1. 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 2. 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.
User Reviews
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Reference:
1. Kim, Jaoon YH, Byung Hoon Jo, and Hyung Joon Cha. "Production of biohydrogen by heterologous expression of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli." Journal of biotechnology 155.3 (2011): 312-319.
UNIQ4eb4281f728818e2-partinfo-00000001-QINU