Project Description

Introduction

With the advancement in the field of synthetic biology, bacteria can be engineered to tackle challenges in various fields such as medicine, energy, and environment. Development of such genetically engineered organisms raises concerns of the accidental releases of engineered microbes from the target host into the external environment, thereby potentially disrupting the balance of the ecosystem. Therefore, it is essential to develop effective and customized kill switches to successfully contain engineered microbes in their designated environment, as shown in figure 1.

Currently, scientists often encounter challenges in the process of designing, constructing, and testing customized kill switches due to the lack of standardized framework to design customized kill switches for different applications.

Agriculture

Bacteria genetically modified to be easily traceable and having improved expression of beneficial traits have been constructed and released in plants in a number of experimental field plots (Amarger, 2002).

Energy

Genetically engineered organisms are used in industrial settings to produce fuels (e.g., Chromatin Inc., Ginkgo Bioworks, LS9 Inc., Solazyme, Verdezyne, and Synthetic Genomics) (Moe-Behrens, Davis & Haynes, 2013)

Medicine

Engineered microbes have been tested to treat diseases like Crohn's disease (Braat et al., 2006) and oral inflammation (mucositis), and can reduce the cost of production of pharmaceuticals (e.g., Ambrx and Amyris) (Moe-Behrens et al., 2013)

Utilities

Engineered organisms can generate renewable chemicals of commercial value (e.g., Genencor, Genomatica Sustainable Chemicals, and Verdezyne) (Moe-Behrens, et. al. 2013)

Environment

Microbes are used to clean up contaminated soil and groundwater. Bioremediation enables the growth of certain microbes that use contaminants as a source of food and energy

Info Tech

Bacteria could be engineered to encrypt messages. Suicide of such bacteria after leaving the contained environment is essential for protection of information. Prevention of bacteria leakage from the research laboratory is also essential to protect intellectual properties (Cai et al., 2015).

Figure 1: Potential applications of engineered bacteria

Our Objective

Team NUSgem aims to develop a methodology (which includes the use of computer aided design and modelling tools, development of toolkit and standardised plasmid with E2 killing protein) to make engineering of customized kill switch easier.

As a proof of concept, we designed and developed a kill switch layered with phosphate and temperature sensors for biocontainment of engineered probiotics.

Our Idea

Our idea is to provide a standardized engineered E. coli plasmid in which the E2 toxin is constitutively expressed with specific, well characterized strength. This plasmid is supposed to be co-transformed with the second plasmid designed by the user with controlled expression of the IM2 protein (antitoxin). As such, users only need to focus on designing and fine-tuning the IM2 (antitoxin) expression to control the kill switch. The IM2 (antitoxin) plasmid can be designed by choosing the suitable sensors and logic that are specific to the application. When the kill switch is activated, IM2 proteins are no longer produced; the accumulation of E2 proteins will kill the bacteria.

Due to time constraints, we focused on developing the methodology and the toolkit for design IM2 (antitoxin) module. The toolkit consists of three major categories: a library of characterized sensor for designed inputs, logic gates to be implemented, and the killing mechanism.

Figure 2: When the sensor detects the 'non-killing' state, high amount of IM2 (anti-toxin) proteins would be produced to sequester the E2 killing protein; enabling the bacteria to survive.

Figure 3: When the sensor is in the 'killing' state, minimal amount of IM2 (anti-toxin) proteins would be produced to sequester the E2 killing protein; hence E2 killing protein accumulates in the cell and killing the cell in the process.

Methodology to Develop IM2 Anti-toxin Module

The IM2 – antitoxin module can be developed by following the steps as shown in figure 4 below:

Figure 4: Framework to develop IM2 anti-toxin module

For detailed explanation of the framework, please click here

Figure 5: NUSgem kill switch Toolkit

NUSgem Kill Switch for Engineered Probiotics

Design of the Kill Switch for Engineered Probiotics

Our kill switch relies on a dual-input (temperature and phosphate) cascade to achieve an OR gate logic. Upon detection of a wastewater environment (low temperature and low phosphate), the expression of anti-toxin IM2 is inhibited, thereby allowing the constitutively produced toxin, endonuclease E2, to destroy the engineered probiotic.

Figure 6: Double plasmids system to control the amount of IM2 (anti-toxin) production and another plasmid to be constitutive for E2 killing protein production.

In the presence of phosphate, PhoR, a sensory histidine kinase, is inhibited. PhoR is activated in low phosphate concentration, phosphorylating PhoB and activating the phoB promoter. TlpA promoter is temperature sensitive (Piraner, Abedi, Moser, Lee-Gosselin & Shapiro, 2016). It is activated when the temperature is above 36 degrees Celsius, upregulating the downstream IM2 production. E2-IM2 is a toxin-antitoxin system. IM2 is an immunity protein that binds to Colicin E2 and inhibit the endonuclease activity.

Figure 7: Truth table for IM2 production

Figure 8: State diagram of the double plasmid system

Probiotics are transported in a phosphate-containing medium and phosphate levels in the gastrointestinal tract are generally high except in the colon (Metcalf et al., 1987). Temperature does not matter in the transportation stage, and will be above 36 degrees Celsius after the probiotics are consumed (human body temperature). IM2 production is only inhibited when both phosphate and temperature levels are low, resulting in killing of the probiotic. Figure 8 represents the states at different timings.

Figure 9: Timing diagram of the double plasmid system

To make sure the circuit will work as expected, we modeled a design and completed our experiments.

Reference

Amarger, N. (2002). Genetically modified bacteria in agriculture. Biochimie, 84(11), 1061-1072. http://dx.doi.org/10.1016/s0300-9084(02)00035-4
Braat, H., Rottiers, P., Hommes, D., Huyghebaert, N., Remaut, E., & Remon, J. et al. (2006). A Phase I Trial With Transgenic Bacteria Expressing Interleukin-10 in Crohn’s Disease. Clinical Gastroenterology And Hepatology, 4(6), 754-759. http://dx.doi.org/10.1016/j.cgh.2006.03.028
Cai, Y., Agmon, N., Choi, W., Ubide, A., Stracquadanio, G., & Caravelli, K. et al. (2015). Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proceedings Of The National Academy Of Sciences, 112(6), 1803-1808. http://dx.doi.org/10.1073/pnas.1424704112
Metcalf, A., Phillips, S., Zinsmeister, A., MacCarty, R., Beart, R., & Wolff, B. (1987). Simplified assessment of segmental colonic transit. Gastroenterology, 92(1), 40-47. http://dx.doi.org/10.1016/0016-5085(87)90837-7
Moe-Behrens, G., Davis, R., & Haynes, K. (2013). Preparing synthetic biology for the world. Frontiers In Microbiology, 4. http://dx.doi.org/10.3389/fmicb.2013.00005
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

Team:NUS Singapore/Description