Part:BBa_K4662042

RhlR with LasB and CFP Level 2 plasmid containing constitutive expressed RhlR level 1 plasmid and inducible LasB promoter with CFP level 1 plasmid.

BBa_K4662028: RhlR is an enzyme that acts as a transcription factor, as soon as the concentration of C4-HSL reaches a certain threshold binds and binds to RhlR. In the bound state RhlR can activate LasB transcription of specific genes.

BBa_K4662038: When LasB is activated it will express CFP.

The two level 1 plasmids are connected with two adapters to form a level 2 plasmids.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 714
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 714
  Illegal NheI site found at 29
  Illegal NheI site found at 52
  Illegal NheI site found at 367
  Illegal NheI site found at 608
  Illegal NotI site found at 856
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 714
  Illegal BamHI site found at 708
  Illegal XhoI site found at 865
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 714
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 714
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

As already mentioned in our contribution to the BBa_R0079 RhlR normally induces the promoter RhlAB. We wanted to increase the iGEM parts collection by confirming that RhlR is also able to induce the LasB promoter (2). This is the consequence of a crosstalk between two different quorum sensing systems. This finding is useful for future teams, since this can be used to finetune the expression of the protein of interest dependent on the quorum sensing signal. (1)

LasB dependent CFP fluoresence

Figure 1: Raw measurement of fluorescence. The untransformed E. coli also has a fluorescent signal at 429 nm excitation

Figure 2: Corrected values for the fluorescent signal of the negative control measured at 429nm excitation.

For testing the RhlR ability to bind to the LasB promoter, we used the level 2 plasmid which contains the RhlR and LasB expressing CFP when induced. We performed a 24h time incubation and measured the fluorescence of CFP over time, to check whether the intensity increases upon adding a different amount of C4-HSL manually to the medium of the bacteria. The untransformed bacteria acted as a negative control.

Evidently seen in figure 1, the fluorescence increases already 2 hours after induction of the system. During the exponential phase, the bacteria are metabolically very active and thus the promoter might be leaking some expression of CFP, as we see in the hours 0-16. After the cells reach saturation, we don’t observe a stronger induction of fluorescence at e.g.: 1 mg/ml C4-HSL compared to 0.01 mg/ml, since the quorum sensing system is a switch like mechanism, resulting in either complete induction or no induction at all (citation). The system is very sensitive and completely induced at 0.01 ug/ml C4-HSL.

Conclusion

The rhl-las quorum sensing hybrid system, we were able to confirm that RhlR is also able to induce the LasB promoter.

Autoinduction of the quorum sensing system

Figure 3: Fluorescent signal at 429 nm excitation of the transformed but uninduced E.coli compared to the transformed and induced E.coli.

To confirm that our E.coli is able to self- induce the production of CFP regulated by the LasB promoter we co-transformed the part with BBa_K4662045 RhlI level 1 assembly into a bacterium. RhlI, which was characterized by us, is a protein that produces a small molecule called N-butanoyl-L-homoserine lactone (C4-HSL).

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 . In figure 3 we can see that the uninduced E.coli has a lower fluorescent signal compared to the E.coli that got self-induced, by C4-Hsl and RhlR.

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.

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.

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