Automating Protein Production Using Multi-chromatic Light
McMaster University iGEM 2015
Presenters: Hamza Qamar, Michael Ranieri, Max Ng, Michael Xie, Karanbir Brar
INTRODUCTION
BACKGROUND
PLASMID DESIGN
Efficient protein expression is an important aspect of pharmaceu3cal manufacturing and biological research. Current expression-­‐isola3on process can be laborious and 3me consuming, and has not been a focus of efficient redesign. RATIONALE
Recently, Tabor et al. demonstrated induc3on of gene expression using mul3chroma3c light and the CcaS/CcaR and C p h 8 / O m p R p h y t o c h r o m e -­‐ r e s p o n s e regulator pairs. We aim to u3lize the Cph8/
OmpR and CcaS/CcaR phytochrome response regulator pairs to create a standardized, biological system capable of expressing protein and autolysing in response to green light and far red light, respec3vely. This system would offer automated protein expression and extrac3on under 3ght control and without the need for further exogenous chemicals.
C c a S / C c a R a n d C p h 8 / O m p R a r e phytochrome-­‐response regulator pairs that can translate an external light s3mulus into an internal cellular response. CcaS (green light) and Cph8 (far red light) ac3vate their his3dine kinase domains in a chromophore-­‐
dependent manner. This results in autophosphoryla3on and phosphotransfer to a response regulator (CcaR or OmpR). The response regulator then binds to a promoter region (PcpcG2 for CcaR or PompC for OmpR) and induces gene expression. 1) Chromophore Plasmid: ho1 and pcyA convert heme into PCB. pBAD promoter, induced by arabinose. METHODS
2) Red-­‐induced Plasmid: C p h 8 i s u n d e r cons3tu3ve expression), GFP under control of PompC . 3) Green-­‐induced Plasmid. T4 holin and endolysin regulate lysis. HARDWARE DESIGN
Holin proteins in conjunc3on with endolysin are a method of causing cell lysis in bacteria. Holin proteins are u3lized by viruses to form holes in the bacterial membrane that result in both membrane depolariza3on and the export of viral endolysin to degrade the pep3doglycan layer. CONCLUSIONS
This system would have important applica3ons in both research and industry, allowing for the op3miza3on and poten3al automa3on of heterologous bacterial protein produc3on. REFERENCES
1.  Tabor, J. J., Levskaya, A., & Voigt, C. A. (2011). Multichromatic control
of gene expression in Escherichia coli. Journal of Molecular Biology,
405(2), 315–324. http://doi.org/10.1016/j.jmb.2010.10.038
2.  Please refer to wiki for remaining references
ACKNOWLEDGEMENTS
We would like to thank our supervisors Alison Cowie, Dr. Kimberly Dej,
Dr. Rosa Da Silva, and Dr. Marie Elliot for contributing lab space,
materials, and their wisdom. We would also like to thank our sponsors,
and the entire McMaster mGEM team for all of their contributions and
hard work.
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All of the designs, constructs, and experiments presented in this poster were performed by the students of the 2015 Hamilton McMaster University team.
Poster and Wiki design by Kushal Panchal.