Difference between revisions of "Part:BBa K3888004"
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<partinfo>BBa_K3888004 short</partinfo> | <partinfo>BBa_K3888004 short</partinfo> | ||
| − | HoxK1 coding gene with E. coli signal sequence | + | HoxK1 coding gene with E. coli signal sequence<br> |
This basic part is used to achieve heterologous expression of small subunit of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli. HoxK1 encodes the small subunit of hydrogenase mentioned above, and together with HoxG1(BBa_K3888003), to form composite part Core Gene HoxG1 and HoxK1(BBa_K3888011). | This basic part is used to achieve heterologous expression of small subunit of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli. HoxK1 encodes the small subunit of hydrogenase mentioned above, and together with HoxG1(BBa_K3888003), to form composite part Core Gene HoxG1 and HoxK1(BBa_K3888011). | ||
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[[File:pET28-pTac-lacO-hydrogenase .png]] | [[File:pET28-pTac-lacO-hydrogenase .png]] | ||
===Figure 1: The Oxygen-tolerant Hydrogenase introduction plasmid constructed, which has a length of 8261bp.=== | ===Figure 1: The Oxygen-tolerant Hydrogenase introduction plasmid constructed, which has a length of 8261bp.=== | ||
HoxK1 encodes the small subunit of oxygen-tolerant hydrogenase, and it's controlled by a Lac operator. | HoxK1 encodes the small subunit of oxygen-tolerant hydrogenase, and it's controlled by a Lac operator. | ||
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| + | ==Applications of BBa_K3888004== | ||
| + | According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows: | ||
| + | [[File:Experiments hydrogenase.png]]<br> | ||
| + | ===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.<br> | ||
| + | The results of electrophoresis are as follows: | ||
| + | |||
| + | [[File:Electrophoresis hydrogenase.png]] | ||
| + | ===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. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
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<partinfo>BBa_K3888004 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3888004 SequenceAndFeatures</partinfo> | ||
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Latest revision as of 17:06, 21 October 2021
HoxK1
HoxK1 coding gene with E. coli signal sequence
This basic part is used to achieve heterologous expression of small subunit of oxygen-tolerant Hydrogenovibrio marinus [NiFe]-hydrogenase in Escherichia coli. HoxK1 encodes the small subunit of hydrogenase mentioned above, and together with HoxG1(BBa_K3888003), to form composite part Core Gene HoxG1 and HoxK1(BBa_K3888011).
Figure 1: The Oxygen-tolerant Hydrogenase introduction plasmid constructed, which has a length of 8261bp.
HoxK1 encodes the small subunit of oxygen-tolerant hydrogenase, and it's controlled by a Lac operator.
Applications of BBa_K3888004
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 940
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]