Team:SJTang/Proof Of Concept
Proof of concept
Overview
Our project is to improve microorganisms to efficiently produce hydrogen, and at the same time, design a complete set of hardware for gas collection and detection in the entire process.
Figure.1 Conceptual drawing of hardware design.
Rhodopseudomonas palustris modification
Rhodopseudomonas palustris is a gram-negative bacterium that can synthesize hydrogen under anaerobic conditions. In order to increase its hydrogen production, we decided to knock out two of its genes: uppE and hupS. UppE is a member of the Upp gene cluster. This gene cluster is responsible for the synthesis of UPP (unipolar polysaccharide), and UppE encodes PHPT (polyisoprenylphosphate hexose-1-phosphate transferase). We believe that deleting this gene can prevent the synthesis of UPP between cells, reduce the appearance of Biofilm, and increase the growth density of R. palustris during fermentation.
Figure.2Upp gene cluster. RPA2750 corresponds to uppE gene.
HupS encodes the small subunit of uptake hydrogenase, which enables R. palustris to oxidize hydrogen and transfer the electrons in the hydrogen to ferredoxin and cytochrome. Deleting the hupS gene can reduce the hydrogen consumption of Rhodopseudomonas palustris during the growth process, thereby assisting the hydrogen biosynthesis process.
Figure.3Uptake hydrogenase gene cluster.
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.3Plasmid 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.5Gene 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.
Carbon tracking bio-reactor
Through there have been much interest, producing hydrogen through the culturing of Rhodopseudomonas palustris faces a series of unique challenges, and it is long hindered by low efficiency. The photobioreactor system that we designed aims to address some of the key issues in bacterial growth, gas collection and product analysis. We believe that cost effectiveness, user friendliness, and flexibility forms is the 3 most important elements in hardware design. So we carried out the design mentioned above.
In general, the entire hardware consists of the following parts: Arduino, driver board, production pathway and analysis pathway. After the hardware is spliced as a whole, the style is as follows:
After debugging the hardware body and code, the whole hardware operation process is as follows: