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OUTLINE

Overview

Shewanella

In our project, Shewanella is responsible for the production of formic acid to maintain this system. It has been reported that overexpression of one important component, ccta (STC), makes a great difference to the efficiency of extracellular electron transport (EET) [1] , which also plays an imperative role in electrosynthesis. As a result, we designed a system that achieves the high expression of this protein. Under the addition of IPTG, the promoter will activate the expression of ccta.

pipeline

pGEX-4T-1 is chosen as it has the promoter required and high popularity in Gram-negative bacteria engineering. According to literature, Shewanella is resistant to ampicillin [2] , leading to a substitution of ampicillin resistant gene for Kanamycin resistant gene.

pGEX-4T-IGEM-1

Limited by condition and budget, we can’t obtain pBBR1MCS-2, thus we need to find a suitable plasmid to integrate cctA.We need to change the resistant gene.Refer to the initial design, we substitute the AmpR gene for KanR gene.

pGEX-4T-IGEM-1

Cyanobacteria

Cyanobacteria are introduced with a plasmid containing the CscB gene to export sucrose from the cell. We got the plasmid from ShanghaiTech_China 2022.

pUC57

Vibrio natriegens

Vibrio natriegens is introduced with the CA (carbonic anhydrase) gene, CARP gene, and Cabp-Chbd gene to fix CO2 and promote mineralization.

pipeline-1 pipeline-2

We selected pETDuet-1 and PACYCDuet-1 as the vectors due to their compatibility. We replaced the original promoter and operator of PACYCDuet-1 with a chitin promoter to initiate mineralization when the chitin concentration in the environment is high. However, the experiment did not produce the expected results, so we reverted to the original promoter and operator, using IPTG induction to express Cabp-Chbd.

pETDuet-1 final 5260碱基对

Table

Shewanella

In order to obtain a stable expression, other components are selected in conformity with another paper [3] .

Part Numbers Name Type Part Description
BBa_K180000 Ptac Promoter A hybrid promoter composed of lac and trp promoter, induced by IPTG.
BBa_K3914035 double terminator rrnb T1T2 Terminator Terminator for bacterial expression.
BBa_B0034 Ribosome binding site Ribosome binding site The sequence for ribosome to bind to.

Vibrio natriegens

Number Name Description
BBa_K4621004 LPMO A whole functional sequence in front of the monooxygenase gene, including its promoter. But we only used the promoter of this part.
BBa_B0029 RBS 1 bp deletion from B0031
BBa_B0030 RBS Strong RBS based on Ron Weiss thesis.
BBa_K2547003 Carbonic anhydrase (csoS3)-His An improved part of carbonic anhydrase (csoS3) from Halothiobacillus neapolitanus which converts the incoming bicarbonate into carbon dioxide in the carboxysome, a step that is essential for CO2 fixation.
BBa_K5397999 CARP Coral acid-rich proteins (CARPs) not only bind Ca2+ stoichiometrically but also precipitate aragonite in seawater at pH levels of 8.2 and 7.6 through electrostatic interactions with protons on bicarbonate ions. Phylogenetic analysis suggests that at least one type of CARP has evolved from a gene fusion event.
BBa_K5397987 CaBP-ChBD CaBP-ChBD significantly promotes carbonate biomineralization on a chitin substrate.
References
[1] Delgado, V. P., Paquete, C. M., Sturm, G., & Gescher, J. (2019). Improvement of the electron transfer rate in Shewanella oneidensis> MR-1 using a tailored periplasmic protein composition. Bioelectrochemistry (Amsterdam, Netherlands), 129, 18–25. https://doi.org/10.1016/j.bioelechem.2019.04.022
[2] Cao, Y., Song, M., Li, F., Li, C., Lin, X., Chen, Y., Chen, Y., Xu, J., Ding, Q., & Song, H. (2019). A Synthetic Plasmid Toolkit for Shewanella oneidensis> MR-1. Frontiers in microbiology, 10, 410. https://doi.org/10.3389/fmicb.2019.00410
[3] 3.Li, F., Li, Y. X., Cao, Y. X., Wang, L., Liu, C. G., Shi, L., & Song, H. (2018). Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis>. Nature communications, 9(1), 3637. https://doi.org/10.1038/s41467-018-05995-8
[4] AHUT_China iGEM Team. (2018). Part: BBa_K2547003. Retrieved from https://parts.igem.org/Part:BBa_K2547003
[5] Cao, Y., Song, M., Li, F., Li, C., Lin, X., Chen, Y., Chen, Y., Xu, J., Ding, Q., & Song, H. (2019). A synthetic plasmid toolkit for Shewanella oneidensis MR-1. Frontiers in Microbiology, 10, 410. https://doi.org/10.3389/fmicb.2019.00410
[6] Delgado, V. P., Paquete, C. M., Sturm, G., & Gescher, J. (2019). Improvement of the electron transfer rate in Shewanella oneidensis MR-1 using a tailored periplasmic protein composition. Bioelectrochemistry (Amsterdam, Netherlands), 129, 18–25. https://doi.org/10.1016/j.bioelechem.2019.04.022
[7] Li, F., Li, Y. X., Cao, Y. X., Wang, L., Liu, C. G., Shi, L., & Song, H. (2018). Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis>. Nature Communications, 9(1), 3637. https://doi.org/10.1038/s41467-018-05995-8
[8] Mass, T., Drake, J. L., Haramaty, L., Kim, J. D., Zelzion, E., Bhattacharya, D., & Falkowski, P. G. (2013). Cloning and characterization of four novel coral acid-rich proteins that precipitate carbonates in vitro. Current Biology, 23(12), 1126-1131. https://doi.org/10.1016/j.cub.2013.05.007
[9] SCUT-China iGEM Team. (2023). Part: BBa_K4621004. Retrieved from https://parts.igem.org/Part:BBa_K4621004
[10] Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K., & Tanaka, H. (1990). Gene cloning of chitinase A1 from Bacillus circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of fibronectin. Journal of Bacteriology, 172(1), 401-409. https://doi.org/10.1128/jb.172.1.401-409.1990
[11] Weiss, R. (n.d.). Part: BBa_B0030. Retrieved from https://parts.igem.org/Part:BBa_B0030
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