Team:BNU-China/kill

Kill Switch

We designed a kill switch to control "Prometheus" and to prevent potential contamination.

One difficulty we face is how to trigger the suicide progress spontaneously at a certain time. In the medium, the bacteria are easily controlled by adding or removing regulatory factors. However, after pouring E. coli into soil, it is hard for us to control. The suicide progress needs to be activated spontaneously. Moreover, the kill switch is supposed to be “off” for a certain time in the soil, so the bacteria will gain enough time to perform its function.

Considering the problems, toxin protein MazF is the best candidate for "Prometheus" to suicide with, as well as for us to restrict the bacterial number under reasonable level.

Background

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.

With regard to mazEF system, we designed the kill switch forE. coli.

Design:

We plan to establish a new E.coli strain without lacI and MazEF system gene to avoid the inference of cell itself. We will test cI background expression level to see if we need to knock out cI.

As is shown in Fig.1, 2, 3, and 4, in "Stable State", before poured into the soil, we culture the bacteria fertilizer in the medium with IPTG. Promoter 1 is a weak constitutive promoter, so lacI is transcribed and translated at a considerate low level. High concentration of IPTG, which inactivates LacI by changing its conformation, could turn on promoter 2. Then, CI can be highly expressed to repress promoter3. At this stage, mazF expression could be inhibited.

Once poured into soil, the bacteria are no longer exposed to that level of IPTG. As a result, LacI with a stabilizing tag takes time to accumulate to the repression threshold of promoter 2. When lacI concentration exceeds the threshold, promoter 2, as well as the expression of cI, get repressed. Time needed to finish this progress is referred to as "memory time". Before lacI reaching the threshold, E. coli takes its own responsibility to deliver Mo. After that, suicide would be triggered.

As shown in Table 1, all the biobricks in the design are from the top 10 most used parts of iGEM to guarantee the feasibility.

Table 1 Biobricks used in the design

In the future, we plan to model the “memory time” and do more wet-experiments to confirm its feasibility. As for the modeling, we will test the expression level of promoter 1, the minimal stand of LacI to repress promoter 2 and the degradation speed of LacI. On the other hand, we will test the background expression of CI after removing IPTG to check if the CI concentration is low enough to open the promoter 3. Then we will strive to make this system more effective.

References

Hazan R, Engelberg-Kulka H. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria. Journal of Cell Science 118:4327-4332

Hazan R, Sat B, Engelberg-Kulka H. Escherichia coli mazEF-Mediated Cell Death Is Triggered by Various Stressful Conditions. Journal of Bacterology 186(11):3663-3669.

Schuster CF, Park JH, Prax M.Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum. Journal of Bacteriology 195:115-125

Lewis D, Le P, Zurla C, Finzi L, Adhya S.Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro. PNAS 108:14807-14812.

Wedler H, Wambutt R. A temperature-sensitive lambda cI repressor functions on a modified operator in yeast cells by masking the TATA element. NCBI 248:499-505.

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The Story of E.coli Prometheus

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