Part:BBa_K2232025

mazEF switch

Encodes a stable non-specific ribonuclease toxin (mazF) and its inhibitory antitoxin (mazE) in Bacillus Subtilis. These genes are used in Bacillus Subtilis to provide a toxin-antitoxin kill switch in various stressful conditions. When expression of both genes is turned off (as both are under contol of same promoter) mazE will be degraded faster than mazF. There is then no inhibiton of mazF, killing the cell. Contains Sucrose sensitive inducer that will only allow coding sequence translation in the presence of sucrose. These genes are used in Bacillus subtilis to provide a toxin-antitoxin kill switch in various stressful conditions. When translation of both genes is turned off by sucrose limitation mazE will be degraded faster than mazF. There is then no inhibiton of mazF, killing the cell.

Improved Uses

Team CLS_CLSG_UK 2020 has adapted this kill switch to increase its efficiency. As well as changing the chassis organism to E. coli given its more widespread use, we also redesigned the workings of the kill switch. Rather than relying on the cell to degrade the MazE quicker than the MazF we have designed a system whereby the production of the MazE is constituitive and under the control of the standard CMV promoter while the MazF is under the control of a hypoxia induced promoter. Assuming that the promoter that controls the production of the toxin has been characterised with the CMV promoter this system can be adapted to any conditions, giving a kill switch that be triggered by any inducible promoter on the registry that has been characterised with CMV and the appropriate changes are made to the RBS to check that their binding strengths are also correct (this can be found in the literature). This system is a large improvement on the original method, it allows for a more long term use for the kill switch, allowing the bacteria to survive with the kill switch inactivated for as long as needed - which the original system doesn't allow for as the constant levels of MazE reached are lower than that of MazF due to the difference in degradation rate. By not relying on the cells breakdown for the kill switch, it can now be used for more long-term, sustainable, bioengineering solutions. The full composite part:BBa_K3588014 details the kill switch or more can be found at https://2020.igem.org/Team:CLS_CLSG_UK/Design

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 363
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

iGEM2017 SZU-China confirmed the function of BBa_K2232025.

We made characterization of this part in our chassis B.subtilis WB800.The sucrose was added in the culture medium when the value of OD₆₀₀ reach stationary phase initially, and the final concentration of sucrose was 20mmol/L. Then we recorded the variation of OD₆₀₀ by interval of 4 hours and the result is shown as follow.

Fig. The OD₆₀₀ of original strain WB800 and recombinant B.subtilis WB800_MazEF recorded by interval of 4 hours.The red arrow represents the adjunction of Sucrose (20mmol/L).

As can be seen from the figure above, the Sucrose-limitation induced kill switch in B.subtilis didn’t show obvious function on our chassis.The reasons may be the concentration of sucrose wasn’t suitable or the degradation of ribonuclease toxin (mazF) is too fast.

References

• Considering the biosafety in our project, we used the original part BBa_K302035 from iGEM10_Newcastle team, which is a suicide switch induced by sucrose.
• Engelberg-Kulka H, Hazan R, Amitai S. mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria[J]. Journal of Cell Science, 2005, 118(19):4327-4332.