Poster in SEED 2017, Vancouver

Improving electron transfer in cytochrome c-expressing Escherichia coli by modulating cytochrome c maturation

Lin Sua,d, Tatsuya Fukushimaa, Moshe Barucha, Jose Cornejoa, Caroline M. Ajo-Franklina,b,c*

Author Affiliations: aMolecular Foundry, bMolecular Biophysics and Integrated Biosciences, and cBiological Systems and Engineering Divisions, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720

dState Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210018, China

*To whom correspondence may be addressed: 1 Cyclotron Rd, Mailstop 67R5115, Berkeley, CA 94720, 510-486-4299, cajo-franklin@lbl.gov

Abstract: Heterologously expressing Shewanella oneidensis MR-1 cytochromes c (cyt c) in Escherichia coli enables cellular states to be monitored and controlled through electron flow. However, the electron flux in E. coli is significantly less than Shewanella oneidensis MR-1, thus limiting the extent of our electronic control. We hypothesize that differences in the cytochrome c maturation (ccm) machinery between these two species may be responsible for the lower expression levels of cyt c and extracellular electron transfer (EET) efficiency. In this research, we constructed random mutations within ccm genes by prone error PCR and screened for increases in cyt c production in E. coli in 96-well plates. Two ccmH mutation strains were shown to exhibit increased cyt c expression. Since these two mutations occurred in the C-terminal domain of ccmH, which has a similar structure of ccmI in the Shewanella oneidensis MR-1, we also constructed a hybrid ccmH comprising the N-terminal domain of Escherichia coli and the C-terminal domain of Shewanella oneidensis MR-1 (ccmHN:ccmI). Indeed, cells from this engineered ccmHN:ccmI strain possess a redder color and higher expression of cyt c. We measured the electron flux out of these modified variants using three-electrode microbial electrochemical experiments and showed that the ccmHN:ccmI strain produces ~40% more current per cell. Thus, modulating maturation of cyt c is not only possible, but can improve electron transfer in synthetic biological systems.

Keywords: ccm machinery, ccm H, extracellular electron transfer

 

AND! Also got 90s to present my work on the stage!!

Good afternoon everyone. I am Lin Su, a graduate student from Dr. Caroline Ajo-Franklin’s group, in the Molecular Foundry, Lawrence Berkeley National Lab. My work is focusing on using synthetic biology to build an electrical connection between microbes and electrodes. By doing that, we can have the access to read or even control the behaviors of the bacteria. While one of the challenges is you need to teach the bacteria to make some complicated proteins called cytochrome c. I have shown in my poster that if you modified how to make the cytochromes, you can get some exciting results in terms of the electric signals. So if you are interested in what we did and how we did it, or you have any suggestion on how to use this connections, please come to my poster. My poster No. is 140. Thank you very much.

 

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