Part:BBa_K4817008:Design

Quorum sensing induced suicide system(low AHLs)

Assembly Compatibility:

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

  Illegal NheI site found at 4029
  Illegal NheI site found at 4052
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1813
  Illegal BamHI site found at 3645
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1263
  Illegal BsaI.rc site found at 3095
  

Design and Properties

Figure 1 pET-28a-luxR-ccdA/ccdB characterization verification line

We introduced the successfully characterized and validated Lux operon (BBa_K407012) from Part3 (We'll cover that in the next section) into the ccdA/ccdB antitoxin-toxin system constructed in Part2 (BBa_K4137006, BBa_P1010 ) upstream, constituting Part0.

Figure 2 Final Logic Circuit Verification

As the stress caused by CcdB to the engineering bacteria will lead to self-cutting of the ccdB gene fragment, we design short-time liquid culture induction experiments， according to the characteristics that AHL can not tolerate high temperature for a long time and is not applicable to be induced in the solid medium. We spread the plates every 1h and plotted the growth curves by the number of colonies in the plates at different times for the verification of the logical line.

Our suicide system inhibited bacterial growth in the absence of AHLs during the first two hours, and the number of colonies in the control group induced by adding AHLs was higher than that in the experimental group without AHLs throughout the incubation process. However, we found the growth of the experimental group and the control group converged to the same after 2-3h of AHL-induced expression, which indicated that the stress caused by CcdB on the engineered bacteria, as well as the excision of the ccdB fragments from the engineered bacteria, occurred in advance. We suspect that the leakage expression of CcdB intensified after the introduction of the quorum-sensing promoter. Therefore, we will continue to explore options to reduce the stress of CcdB on the host bacterium during the subsequent improvement of the project.

Source

E.coli

References

[1] Liang Y, Pan Y, Li Q, Wu B, Hu M. RNA-seq-based transcriptomic analysis of AHL-induced biofilm and pyocyanin inhibition in Pseudomonas aeruginosa by Lactobacillus brevis. Int Microbiol. 2022 Aug;25(3):447-456.