Team:Michigan/Description
Project Description
The Project
This year, the Michigan Synthetic Biology team explored the applications of temperature-based cellular modulation. We engineered a temperature-controlled bacterial killswitch, designed to prevent survival of cells outside of a desired range, specifically one that is associated with typical laboratory working conditions, as a safety precaution in lab settings. The genetic switch leverages a temperature-dependent promoter to trigger self-lysis in cells. The switch design incorporates a holin/antiholin lysis mechanism, that is initiated if the environmental temperature falls below 34C for a prolonged length of time. As proof of concept, we attempted to construct the kill switch in E. coli. We designed experiments to test the rate of cell death at variable temperatures after growth at 37C.
Why?
We set out to create a plasmid that could be used as an additional safety precaution when working in laboratory environments with dangerous pathogenic bacteria. The project was initially inspired by the library of tunable temperature-sensitive promoters reported in Tunable thermal bioswitches for in vivo control of microbial therapeutics (Piraner et. al., 2016). Other iGEM teams in the past have attempted to develop kill-switches, but they all depended on chemical treatment to trigger self lysis (Knight, 2015). This presents practical limitations as chemical treatment of escaped or uncontrolled bacterium may prove impossible or inefficient. Our solution was to use temperature as a trigger for the self-lysis mechanism. Bacteria with our proposed plasmid will self-destruct when outside their designated, controlled environment. The switch could also prevent the transfer of antibiotic resistance from escaped bacterium to pathogenic strains, decreasing the rate at which antibiotic resistance spreads.
Although we developed our construct in E. coli as a proof of concept, we envision the final product being incorporated into the genome of research strains species, such as S. aureus. This strain can be antibiotic resistant and is known to be susceptible to the holin/endolysin system our switch employs, as seen in Phages of Staphylococcus aureus and their impact on host evolution (Xia and Wolz, 2014).
For example, suppose that a team of researchers was conducting a study that was testing the effectiveness of several novel antibiotic compounds to kill methicillin-resistant Staphylococcus aureus (MRSA). If improperly disposed of, or tracked out by a researcher, these bacteria could escape into the outside environment and cause infection among civilians. More concerning is the possibility that environmental bacteria may obtain the methicillin resistance gene by horizontal gene transfer. If these bacteria contained our proposed kill switch, they would perish soon after leaving the temperature-controlled environment, greatly reducing the chance of infection or resistance plasmid transfer.
Parts
Our proposed switch design uses a temperature sensitive genetic circuit that relies on the temperature dependent repressor TlpA36, and three proteins isolated from the T4 bacteriophage; T4 endolysin, T4 holin, and T4 antiholin.
TlpA36
It is a temperature-dependent promoter/repressor system first created and characterized in Tunable thermal bioswitches for in vivo control of microbial therapeutics (Piraner et. al. 2016). It is a mutant version of a repressor found naturally in Salmonella typhlmurium. The TlpA36 protein will strongly repress any gene downstream of the TlpA promoter, but will undergo a drastic deformation in the narrow temperature range of 37C to 45C. Within this range, the gene downstream of the promoter will be expressed since it is not being repressed by the deformed TlpA36. The repressor protein itself is under the control of a sigma 70 constitutive promoter making its expression independent of temperature.
T4 Endolysin
It is a lysozyme that lyses a bacterial cell from the inside. It does this by degrading the peptidoglycan layer of the cell wall, exposing the bacteria to the extracellular environment and killing it. Its activity can be improved by the presence of T4 holin, a protein that opens pores in the cell membrane and wall, allowing lysozymes such as endolysin to degrade the cell wall faster. T4 holin is repressed in the presence of T4 antiholin, which binds to T4 holin’s soluble domain. This in turn reduces T4 endolysin’s potency.
T4 Holin
It is a protein isolated from the T4 bacteriophage that lyses a bacterial cell from the inside. T4 holin opens pores in the cell membrane and wall, allowing lysozymes to degrade the cell.
T4 Antiholin
It is a protein isolated from the T4 bacteriophage that prevents cell lysis due to T4 holin by binding to T4 holin’s soluble domain. In nature T-even phages produce T4 antiholin to delay the destruction of their host cell.
How It Works
Our design places T4 antiholin under the control of the TlpA36 promoter. T4 endolysin and T4 holin are expressed constitutively. We cloned three different versions of the construct, each with different strength for this promoter in order to leverage the kinetics between Holin/Endolysin and Antiholin. We used the T, JJ, L promoters characterized in Tuning genetic control through promoter engineering (Alper, Fischer, Nevoigt & Stephanopoulos, 2005). At temperatures below 37C, TlpA36 represses T4 antiholin expression, allowing the constitutively expressed T4 holin and T4 endolysin to lyse the cell and preventing its ‘escape’ into the environment.
At 37C however, TlpA36 stops repressing T4 antiholin, allowing it to prevent T4 holin and T4 endolysin from lysing the cell.
References:
Alper, H., Fischer, C., Nevoigt, E., & Stephanopoulos, G. (2005). Tuning genetic control through promoter engineering. Proceedings Of The National Academy Of Sciences, 102(36), 12678-12683. http://dx.doi.org/10.1073/pnas.0504604102
Knight, H. (2015). “Kill switches” shut down engineered bacteria. MIT News. Retrieved 1 November 2017, from http://news.mit.edu/2015/kill-switches-shut-down-engineered-bacteria-1211
Piraner, D., Abedi, M., Moser, B., Lee-Gosselin, A., & Shapiro, M. (2016). Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nature Chemical Biology, 13(1), 75-80. http://dx.doi.org/10.1038/nchembio.2233
Xia, G., & Wolz, C. (2014). Phages of Staphylococcus aureus and their impact on host evolution. Infection, Genetics And Evolution, 21, 593-601. http://dx.doi.org/10.1016/j.meegid.2013.04.022
