Team:SJTang/Results
Overview
Overall, we successfully synthesized I-HTCC and achieved [Ni-Fe] Hydrogenase expression in E. coli. In the part of Rhodopseudomonas palustris, we conducted various investigations on the newly introduced iGEM chassis bacteria and tried to knock out genes. However, in the end, due to some unknown reasons, we were not able to successfully achieve hydrogen synthesis using modified Escherichia coli or Rhodopseudomonas palustris.
1. E. coli Hydrogen Production
1.1 I-HTCC Production
We prepared the I-HTCC material according to the method in the reference. After hydrothermal reaction, filtration, vacuum drying and grinding, the finished products are as follows:
Figure.1I-HTCC finished product. Left: the powder obtained after grinding; Right: IHTCC finished product stored in deionized water.
1.2 Hydrogenase Production
According to the literature, we synthesized the [Ni-Fe] Hydrogenase expression plasmid. The results are as follows:
Figure.2Hydrogenase 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.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.
1.3 Hydrogen Production
After completing the preparation of IHTCC and the induced expression of hydrogenase, we tried to test the combination of the two. Since I-HTCC is negatively charged, after using PAH(Polyallylamine hydrochloride) to make the surface of E. coli positively charged, the two are mixed. The status of the bacteria obtained after mixing is as follows:
Figure.4I-HTCC adding to E. coli. Left: I-HTCC@E. coli, right: I-HTCC with E. coli.
Since I-HTCC is negatively charged and the PAH treated E. coli is positively charged, the two can bind to each other under the action of electrostatic force (I-HTCC@E. coli). The untreated Escherichia coli cannot combine well with I-HTCC, and the two will have an obvious layered state after centrifugation (I-HTCC with E. coli). The results in the above figure show that the E. coli we processed can bind well to the I-HTCC we prepared.
After that, we used the processed E. coli to conduct a gas production fermentation experiment. However, the results showed that no hydrogen was produced.
2. R. palustris Hydrogen Production
2.1 Gene Knock-out for R. palustris
In order to achieve gene knockout, we used pK18mobsacB-Gm plasmid as a knockout vector. In addition, in order to achieve knockout, we selected about 1000bp sequences in the upstream and downstream of UppE and HupS respectively for design, amplified from the Rhodopseudomonas palustris CGA009 by PCR, and constructed knockout plasmids by the Gibson method (see the Design section for principles) ). The results of plasmid design and construction are as follows:
Figure.5Plasmid Design and Agarose Gel Result. Left: UppE Plasmid Design. Middle: HupS Plasmid Design. Right: Agarose gel result (0.8%).
After the plasmid construction is completed, we use the method of electrotransformation to transfer the plasmid into the cell. Afterwards, colonies in which the plasmid was successfully integrated into the chromosome were screened by the gentamicin resistance gene on the vector. After that, the medium containing 10% sucrose was used for secondary screening to facilitate the excision of the inserted plasmid sequence in the double exchange event. Finally, the selected individuals are amplified and cultured, and the genome is extracted and verified by PCR. In order to advance the progress of the experiment, the first round of knockout experiments were carried out separately, and the knockout results are as follows:
Figure.6Gene knockout design and result. Left: Test Design. Right:HupS Test Result.
Due to the time of the project, we completed the knockout of HupS, but the knockout screening of UppE was not successful. In the end, we failed to complete the knockout of UppE.
2.2 Hydrogen Production with R. palustris
We cultivated Rhodopseudomonas palustris to a certain concentration and then inoculate it into a culture flask for airtight fermentation. However, due to some reasons that we don't know, no hydrogen yield was observed in the end.
Figure.7R. palustris in YPMOPS medium.
Figure.8R. palustris with our Hardware.