Difference between revisions of "Team:Hamilton McMaster/Background"

Background

  Numerous methods for bacterial lysis exist, spanning a wide range of mechanisms such as chemically induced lysis methods (eg. detergents, cell lysis solutions), biologically induced methods (eg. lysozyme) and mechanically induced lysis methods (French press, sonication)¹. While these techniques are valuable and still widely used, some can have inherent weaknesses such as the possibility of denaturing proteins of interest. Many of these techniques also require an operator to be present or would likely require expensive machinery for automation. In nature, viruses have also evolved bacterial cell lysis methods as an integral part of their replication cycles. One of the common methods utilized by viruses is a holin/endolysin system in order to achieve bacterial cell lysis upon completion of viral reproduction within the bacterial cell. This system relies on two major components: an endolysin such as T4 phage endolysin that has muralytic activity and degrades the bacterial peptidoglycan layer, and a holin/antiholin pair that operate to form holes in the bacterial inner membrane upon an environmental cue. The pore formation results in membrane depolarization and the export of endolysin where it can then access the peptidoglycan and degrade it, thereby removing a main source of structural integrity for the cell^3,4. Apart from the various membranes and cell wall, this method of lysis leaves most internal structures such as cytosolic proteins intact, leaves the solution free of contamination from chemical inducers of lysis⁵. This system is therefore open to adaptation by synthetic biologists to create non-denaturing lysis systems that can be controlled through genetic cues.

  Construction and control of gene systems that can be manipulated via external stimuli have been heavily studied and utilized by synthetic biologists. One such example was demonstrated by Tabor et al. in 2014² and involved the use of naturally-occurring phytochromes and their associated chromophores to create gene systems that can be controlled by multichromatic light. Phytochromes are proteins that contain an N-terminal region that functions as a photoreceptor using a chromophore molecule as a cofactor, as well as a C-terminal region that functions as a histidine kinase⁶. Activation of the photoreceptor portion by light catalyzes a conformational change in the protein that leads to autophosphorylation by the histidine kinase region^6,7. The autophosphorylated phytochrome can then activate a response regulator protein that will activate the expression of genes downstream of their respective DNA binding sites^6,7. The phytochromes allow for transcriptional modulation based upon light states outside of the cell, which can be controlled quickly and with ease. Multiple types of phytochromes have been discovered and characterized to date, including those that are able to sense green/red light as well as red/far red light^6,7.

Bibliography

1. Harrison, S. T. L. (1991). Bacterial cell disruption: A key unit operation in the recovery of intracellular products. Biotechnology Advances,9(2), 217–240. http://doi.org/10.1016/0734-9750(91)90005-G
2. 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
3. Wang, I.-N., Smith, D. L., & Young, R. (2000). Holins: The Protein Clocks of Bacteriophage Infections. Annual Review of Microbiology,54(1), 799–825. http://doi.org/10.1146/annurev.micro.54.1.799
4. Moussa, S. H., Kuznetsov, V., Tran, T. A. T., Sacchettini, J. C., & Young, R. (2012). Protein determinants of phage T4 lysis inhibition.Protein Science : A Publication of the Protein Society, 21(4), 571–582. http://doi.org/10.1002/pro.2042
5. Miyake, K., Abe, K., Ferri, S., Nakajima, M., Nakamura, M., Yoshida, W., … Sode, K. (2014). A green-light inducible lytic system for cyanobacterial cells.Biotechnology for Biofuels,7(1), 56. http://doi.org/10.1186/1754-6834-7-56
6. Rockwell, N. C., Su, Y.-S., & Lagarias, J. C. (2006). PHYTOCHOME STRUCTURE AND SIGNALING MECHANISMS. Annual Review of Plant Biology, 57, 837–858. http://doi.org/10.1146/annurev.arplant.56.032604.144208
7. Hirose, Y., Shimada, T., Narikawa, R., Katayama, M., & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences of the United States of America, 105(28), 9528–9533. http://doi.org/10.1073/pnas.0801826105

Our Rationale

 Our project aims to generate an E. coli expression system that can be induced to overexpress a protein of interest simply by exposure to a specific wavelength of light. Once expressed, another wavelength of light will be used to induce bacterial cell autolysis, releasing proteins into the solution for isolation. The overall goal of this project is to consolidate the time consuming process of protein expression and generate an easy automated process. If successful, laboratories and manufacturers will not need to physically add chemical inducers or utilize expensive mechanical equipment to achieve lysis. Furthermore, the overall system has the potential to be automated for relatively low cost. Achieving this will enhance the efficiency and accessibility of protein expression and isolation both in research and production settings.

 To accomplish this we will incorporate the theory behind multichromatic control of gene expression and the holin-endolysin system. The system will consist of three plasmids, a chromophore plasmid that contains the chromophore necessary for activation of light sensitive transcription factors so that the systems can be managed in the presence of light. The red plasmid will contain the protein of interest downstream of a promoter that is induced by red light and the green plasmid will contain the holin-endolysin dual system downstream of a promoter that is induced by green light.