Team:BHSF ND/Project

Safety

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Description
Leakage
Bistable System
Recombinase
Toxin
Hardware
Demonstration
Attributions
Measurement

Overview

To achieve the goal of Digitalizing a bistable module enabling conditional suicide of engineered bacteria, our project is separated into three main digitalized modules: An irreversible recombinase, the bistable module, and the toxin. To merely the experiment, we use XyIs/Pm and Arac/pBAD as the inducible promoter module that represents the environmental input of the factory’s culture. By combining the three digitalized module with the inducible promoter, our complete design is described in the following graph.

Generally, after putting into the working condition, the recombinase is switched, and the suicide module is prepared. In working condition, bacteria functions, and the potential damage of leakage is prevented by the mutual inhibition of the bistable module. However, an external change after the stealing removes the inhibition, and, due to the switch initially caused by the recombinase, the expression of the toxin destroys the engineered bacteria.

Complete design

Leakage:

Through our design and experimental validation, we demonstrate the existence of leaks and the need to address leaks. We tried to use a more efficient promoter, but it didn't solve the leak. The leak-related experiment drove us to design an efficient zero-leakage module to put intoxin and ultimately form the design of the suicide system.

Bistable module:

Through our design and experimental analysis, our bistable finally achieved the desired result: a state of zero leakage through mutual inhibition. This way we form a zero-and-one-converted module.

After mathematical modeling guidelines and standard and modular operations, we finally digitalize our bistable system. Its good experimental results ensure the scientificity, feasibility, and efficiency of our overall design. The basic characteristics of preventing leakage also allow us to put toxin to complete the construction of our complete suicide system.

Our digitalized bistable module can be used efficiently in other synthetic biology and related iGEM projects. This zero-expression leakage effect will effectively promote the development of synthetic biology engineering.

Recombinase:

Our recombinases can effectively invert the in-between promoter under our design and experimental validation. This feature works well as a reactor for changes in the external environment. It permanently changes the sequence of genes and thus the direction of gene expression, which implements our goal of connecting two bistable system and exerting permanent effect on the circuit.

The expression of RFP corresponds with the “0” state when applied to our circuit, where the bacterium hasn’t entered its working condition; while the expression of GFP after P1 has been reversed corresponds with the “standby” state in our circuit. That’s the reason why we improved the expression leakage of the irreversible recombinase in order to ensure that no leaked recombinase is present in inducer’s presence which may accidentally initiates the expression of toxin.

Toxin:

After our design and experimental verification, we proved the availability of toxin in the suicide system. Under the careful reading of iGEM security requirements, we ensure the scientificity, usability and efficiency of the selected toxin. After comparison and screening, we selected the most efficient toxin, Kid, as part of our final conditional suicide system.