Part:BBa_K4662044

RhlI, N-Butyrylhomoserine lactone

RhlI is a protein that produces a small molecule called N-butanoyl-L-homoserine lactone (C4-HSL). This small molecule acts as a signalling molecule and has been described to be well diffusible (1)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 136
  Illegal PstI site found at 574
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 136
  Illegal PstI site found at 574
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 136
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 136
  Illegal PstI site found at 574
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 136
  Illegal PstI site found at 574
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Recombinant production of C4-HSL by RhlI was tested using FIA-MS.

Figure 1: FIA-MS analysis. The intensity is measured at 170 m/z, which corresponds to the deprotonated C4-HSL molecule. A3 = supernatant of untransformed E. coli, B3 = supernatant of engineered E. coli, C1 = extracted cell pellet of untransformed bacterium, D1 = extracted cell pellet of engineered E. coli).

The fact that some signal in the negative control is visible, might be due to the fact that some bigger molecules fragmented during mass spectrometry or a dimerization of two molecules resulted in the same molecular mass. This data shows clearly that the molecule of interest is produced and secreted successfully.

Conclusion Although we could not quantify the concentration of the molecule, we were able to see a clear difference between the negative control and our engineered E. coli.

Autoinduction of the quorum sensing system

Figure 2: Fluorescent signal at 429 nm excitation of the transformed but uninduced E.coli compared to the transformed and induced E.coli.

Testing the capacity of RhlI as an autoinducing molecule, was tested with a co-transformation to clone Level 2 RhlR and LasB with CFP into one bacterium BBa_K4662042 . With the CFP output, we measured the fluorescent signal over time to confirm that the CFP signal increases as soon as the population density of the bacteria increases simultaneously with their production of the C4-HSL molecule.

Conclusion Our E.coli was able to produce the auto-induction molecule itself and after diffusion and binding to RhlR, the LasB controlled CFP expression got activated. We were able to complete characterization of our rhl-las quorum sensing hybrid system.

Usage and Biology

Our Rhl-Las quorum sensing hybrid system can be easily adapted by simply exchanging the CFP moiety to the protein of interest.

References

• [1] Yates Edwin et. al, N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa (2002) https://journals.asm.org/doi/abs/10.1128/iai.70.10.5635-5646.2002

Remark

Specific protocols for assembly and testing can be found on our wiki https://2023.igem.wiki/uzurich/experiments

If any questions arise or additional information is needed, we can provide a more extensive list of experiments that we can share if required.