We designed a sterilization switch to control the "Engineering Bacteria Nissle1917" and prevent potential contamination.
One of the difficulties we face is how engineering bacteria that escape to the in vitro environment spontaneously trigger suicide. We considered the differences between the intestinal and in vitro environments and noted that one of the most obvious variables was the difference in oxygen concentration, which noted the sensitivity of the phyb promoter to changes in oxygen concentration. In the medium, bacteria are easily controlled by adding or removing regulatory factors. However, it is difficult to control E.coli after it has entered the environment. The suicide process needs to start spontaneously. In addition, the kill switch should be "off" in the in vitro environment for a period of time, so bacteria will get enough time to perform their functions.
Given these issues, the toxin protein MazF is the best candidate for engineered bacterial suicide. And the phyb promoter helps the engineer complete the switch between the "on" and "off" states of the suicide switch.
MazEF is a toxin-antitoxin module comprising mazE and mazF locating on E.coli and other pathogens' chromosome (Hanna et al, 2005). The protein product of mazF expression is a stable toxin, while mazE a labile MazF antitoxin (Hazan et al, 2004; Schusteret al., 2013). MazF is a sequence-specific mRNA endo-ribonuclease that, when triggered in response to various stresses initiates programmed cell death. The mazEF-mediated death pathway initially acts as a defense mechanism to prevent the spread of bacterial phage infection, allowing bacterial populations to behave like multicellular organisms.
Figure. 1 A schematic representation of the E.colimazEF-mediated cell death pathway (Hanna et al, 2005)
Phyb，the promoter of hybO gene which is one of three E.coli> hydrogenases synthesized in response to different physiological conditions. HYD2 is involved in hydrogen uptake.
With the sensitive perception of oxygen concentration changes in the phyb promoter, the suicide effect was achieved.
The suicide process can be divided into two parts:
First, under oxygen-free conditions, bacteria will meet lactose, because lactose metabolite isolactose is an inducer of lac manipulators, it can be combined in the inhibitor protein variant At the site, the conformation changes, destroys the affinity between the inhibitor protein and the manipulative gene, cannot bind to the manipulative gene, so the RNA polymerase binds to the promoter, and successfully transcribes the structural gene by manipulating the gene, expressing MazF, At the same time in the intestinal environment under low oxygen conditions, the phyb promoter is energetic, expressing the MazE gene, toxins and antitoxins cancel each other out, engineering bacteria survive.
Figure. 2 Design
Second, under aerobic conditions, the phyb promoter is immediately inactivated, at which point the antitoxin gene is no longer expressed, the suicide system only expresses MazF, once the concentration of the toxin protein MazF reaches a critical value, it will induce apoptosis, bacteria will die.
Here is the plasmid map:
Figure. 3 POxygen_suicide(Lac-mazF-T1-lac-phyb-mazE-T1)
MazEF, phyb are derived from E.coli itself, will avoid the risk of foreign genes interfering with the engineered bacteria themselves. We used the anaerobic glove box to create an oxygen-free environment, simulating the intestinal environment in which the phyb promoter works normally, producing MazE, neutralizing with mazF toxins and allowing the normal growth and metabolism of engineered bacteria.
Using the Gibson assembly method to assemble 5 genes, and successfully obtained the built plasmid, run glue verification as shown in the following figure, the length of about 4.7kb, in line with the plasmid length.
Figure. 4 Gel.Doc.pOxygen_suicide
Figure. 5 The engineered bacteria are cultured under oxygen-free conditions
Nissle 1917, in which the plasmid was successfully introduced, was cultured in glove box. After constantly groping for reaction conditions, it was found that Nissle 1917 grew very slowly in an oxygen-free environment, requiring 48-72h growth in order to see the obvious colony of the crucible in the aerobic air environment, it is not possible to observe obvious traces of engineering bacteria growth, which can indicate the success of the suicide system.
Figure. 6 The engineered bacteria are cultured under oxygen-free conditions
 Nikolic, N., Bergmiller, T., Vandervelde, A., Albanese, T. G., Gelens, L., & Moll, I. (2018). Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations. Nucleic acids research, 46(6), 2918–2931. https://doi.org/10.1093/nar/gky079
 Engelberg-Kulka, H., Hazan, R., & Amitai, S. (2005). mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of cell science, 118(Pt 19), 4327–4332. https://doi.org/10.1242/jcs.02619
 Hazan R, Sat B, Engelberg-Kulka H. Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. Journal of Bacteriology. 2004 Jun;186(11):3663-3669. DOI: 10.1128/jb.186.11.3663-3669.2004. PMID: 15150257; PMCID: PMC415763.