Part:BBa_K4239011

fiatlux operon and the axe-txe toxin/antitoxin system

Description

BBa K4239011 is a combination of the fiatlux (BBa_K4239008) operon and the toxin/antitoxin gene axe/txe (BBa_K42390010).

Axe/txe is a toxin/antitoxin system is made to be used on the same plasmid than an interest gene (the fiatlux operon, in our project). This method of selection ensures that all the bacteria present on the plant have the plasmid, and can hence emit light. Toxins have a longer life span in cells than antitoxins, so if the bacteria lose their plasmid, the toxin is no longer destroyed by the antitoxin. This results in the death of the bacteria, thus assuring the death of all fiatlux non-carriers. As a consequence, by involving a selective pressure, the toxin/antitoxin system increases the stability of the plasmid in the bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 428
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 441
  Illegal NheI site found at 464
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 5776
  Illegal BamHI site found at 484
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 428
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 428
  Illegal AgeI site found at 3427
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1800
  Illegal SapI site found at 3946
  

Construction

The system was inserted in pSEVA531-fiatlux, which was digested, before the fiatlux operon. On the one hand, axe/txe was inserted in the opened plasmid thanks to a TEDA cloning. E.coli DH5α were transformed by both plasmids. After verification thanks to a PCR on colonies, we can conclude that only the plasmid pSEVA53-fiatlux-Axe/Txe worked. In order to make sure that our transformation with pSEVA53-fiatlux-Axe/Txe worked, we also did a restriction map, which showed coherent results.

References

Gregor C, Gwosch KC, Sahl SJ, Hell SW. Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc Natl Acad Sci U S A. 2018 Jan 30;115(5):962-967. doi: 10.1073/pnas.1715946115. Epub 2018 Jan 16. PMID: 29339494; PMCID: PMC5798359.