Team:SJTang/Contribution
Our contribution to iGEM can be classified into two parts:
introducing a new chassis for iGEM & adding information on an existing part.
1. Introducing a new chassis for iGEM
Our team introduced a new chassis for iGEM this year: Rhodopseudomonas palustris. Rhodopseudomonas palustris is a facultative aerobic bacteria. It can grow anaerobic in the light, or grow aerobic in the dark. R. palustris is rod-shaped, slightly curved, and belongs to Gram-negative bacteria. In the anaerobic state, the cell culture fluid initially presents a light red color, and finally turns dark red; in the aerobic state, it is colorless or slightly pink. [1]
Figure.1Photo of R. palustris after Gram stain.
R. palustris, as a typical hydrogen-producing strain, has been mainly reported in articles in recent years that it can grow in an anaerobic state. The growth of R. palustris in liquid media can be measured using OD660nm. Existing articles show that in the YPMOPS medium(Yeast Extract 3g/L, Peptone 3g/L, MOPS 10mM) under anaerobic conditions, R. palustris can reach OD660nm=0.8 at about 200 hours [2]. However, such a growth rate obviously cannot meet the research needs of synthetic biology. After our attempts, we found that after improving the medium, R. palustris can grow rapidly under aerobic conditions.
Figure.2Aerobic growth rate of R. palustris. Sodium succinate used here is 10 mM.
The results showed that after adding 10mM Sodium succinate to YPMOPS medium, R. palustris can reach OD660nm=0.4 after 48 hours, and after further enhancing the nutrient substances in the medium, R. palustris can reach OD660nm=1.0 after 48 hours. This method can greatly shorten the cultivation time of R. palustris.
2. Adding information on an existing part
We also added some information on an existing part. Part BBa K1958001 was uploaded by 2016 Nanjing-China.
In order to realize hydrogen production, we conducted in-depth investigations on hydrogenase. After consulting the relevant literature, we found that the first 132 bp in the HyaA gene sequence encodes the signal sequence of the HyaA gene. This part of the sequence helps the small subunit of Hydrogenase locate on the cell membrane.
Figure.3The signal sequence of HyaA.
Therefore, when our team introduced the exogenous hydrogenase's subunit HoxK1 from Hydrogenovibrio marinus MH-110, we replaced the signal peptide of HoxK1 with the signal sequence of HyaA for correct expression in E. coli. For more details about HoxK1, please search "BBa_K3888011".
We record this information here, mainly to remind the subsequent team that when performing heterologous expression of HyaA or introducing other Hydrogenase small subunit genes into E. coli, they must remember to modify the signal sequence. This is very important for the expression of these proteins.
3. More attempts in molecular cloning of anaerobic bacteria
For the anaerobic bacteria such as Rhodopseudomonas palustris, the traditional method is to use an anaerobic incubator for cultivation, and used E. coli S-17 for conjugation. In this project, we tried rapid cultivation under aerobic conditions and electrotransformation. And, we used pK18mobSacB to successfully knock out the gene in Rhodopseudomonas palustris. Go to Protocols for more details.
References:
1. Boone, D. R., Garrity, G. & Castenholz, R. W. Bergey's Manual of Systematic Bacteriology: Volume One : The Archaea and the Deeply Branching and Phototrophic Bacteria. (Springer New York, 2011).
2. Laing, Ruth Mary Louise. Development of Rhodopseudomonas palustris as a chassis for biotechnological applications. Diss. University of Cambridge, 2018.