Team:BABS UNSW Australia/project/containment

Introduction

Due to the nature of our project, having several measures in place to ensure containment and biosafety is very important. We strongly felt that we should not experiment with introducing the invasion-listeriolysin devices into bacterial hosts until we had developed well characterised and effective safety checks. We focused on two key control measures: the Cre-lox recombination system which would delete genes encoding invasive proteins after entry into a cell, and a toxin-antitoxin system to contain bacteria to specific inducing agar plates and prevent horizontal gene transfer.

The Cre-Lox System [1]

The Cre-lox system is an efficient method for imparting deletions, insertions and translocations at specific sites on a genome. These sites are described by a Lox sequence which consists of a 13bp recognition region followed by an 8bp spacer and then closed by another 13bp recognition sequence. Cre recombinase will recognise two of these sites and effect a recombination event between them. The outcome is dependent on the sequence orientation. This can be seen in the diagram below.

Why?

Once our endosymbiont is inside the host there is no longer a need for it to possess any potential for invasion. To remove the hazardous genes we set up a Cre-Lox system that would not only delete the invasive genes, but also the Cre-lox system itself. This arrangement can be seen in the pHlow plasmid. This is not just a safety mechanism. By removing all of the material it significantly decreases the energetic burden placed on the host cell and should therefore increase the endosymbionts chance of survival.

Future Work

In theory, our Cre-Lox system should remove itself given a long enough period of time. We designed an experiment to test this self-fluxing system to investigate how long it would take. The part we designed can be seen below. A fairly standard ONPG/ONP assay could then be used to measure the deletion time. Due to time restraints we were unfortunately unable to build the part and conduct the assay.

References

[1] Hoess, R. H., & Abremski, K. (1990). The Cre-lox recombination system. InNucleic Acids and Molecular Biology 4 (pp. 99-109). Springer Berlin Heidelberg.

The Toxin-Antitoxin System

The Toxin-antitoxin system can be looked at as a classical poison and an antidote - that is one protein will kill the cell, but the presence of the other protein will counteract it.

Why?

The toxin-antitoxin system that we are using was biobricked by Team Washington (iGEM 2010) and consists of the Tsi2 antitoxin and the Tse2 toxin and was isolated from Pseudomonas aeruginosa. Internal expression of Tse2 causes death in both eukaryotes and prokaryotes - however when co-expressed with Tsi2 a complex is formed that prevents this [1]. In P. aeruginosa, the toxin is a substrate for the Type VI secretion system and can be secreted into gram-negative cells. Meanwhile, Tsi2 cannot be secreted and remains inside the bacteria. This allows production of an otherwise toxic metabolite. E. coli does not contain the Type VI secretion system, and hence both the toxin and antitoxin remain inside the cell. [2,3]

Typically both the toxin and antitoxin are expressed on the same plasmid and the longer half life of the toxin will lead to cell death if the plasmid is lost. We have instead taken an innovative and novel approach with the toxin and antitoxin under different promoters on different plasmids.

The pHlow plasmid contains contains a constitutively expressed Tse2 toxin (under the CP44 promoter) and a secondary plasmid contains the IPTG- inducible antitoxin, Tsi2.

There is a twofold benefit to this toxin-antitoxin system:

  • It prevents the escape of the recombinant bacteria from the lab. If the bacteria escapes there will be no IPTG present in its external environment, leading to no expression of Tsi2 and ultimately bacterial death. This ensures that the bacteria can only grow in the laboratory environment.

  • Additionally this also prevents horizontal gene transfer (HGT) between bacteria. If a bacteria receives the pHlow plasmid (either by conjugation or uptake of the naked plasmid) but not the antitoxin plasmid the expression of the Tse2 will lead to cell death. This will prevent the spread of the pHlow system to other bacterial populations.

Design

There are several design considerations involved in the Toxin-antitoxin system, with the main being the naturally availability of alternative inducers. While IPTG does not occur naturally and is a much more potent inducer of the IPTG promoter, lactose can also induce expression. If lactose is present in the external environment (and the bacteria are able to take it up) Tsi2 could be expressed, leading to recombinant bacteria surviving outside of a controlled laboratory environment. Using a more tightly regulated inducible promoter, or a purely artificial promoter could further ensure that no recombinant bacteria could escape from the laboratory.

On top of this, a new containment system would have to be found if the bacteria were living in mammalian cells.

References

[1] Hood RD, Singh P, Hsu F, Güvener T, Carl MA, Trinidad RR, Silverman JM, Ohlson BB, Hicks KG, Plemel RL, Li M, Schwarz S, Wang WY, Merz AJ, Goodlett DR, Mougous JD. A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe. 2010 Jan 21;7(1):25-37. PMID: 20114026

[2] Filloux, Alain, Hachani, Abderrahman, Bleves, Sophie The bacterial type VI secretion machine: yet another player for protein transport across membranes Microbiology 2008 154: 1570-1583

[3] Li, M., Le Trong, I., Carl, M. A., Larson, E. T., Chou, S., De Leon, J. A., ... & Mougous, J. D. (2012). Structural basis for type VI secretion effector recognition by a cognate immunity protein. PLoS Pathog, 8(4), e1002613.