Part:BBa_R0079

Promoter (LasR & PAI regulated)

Binding region for LasR protein (positive regulation)

Usage and Biology

Abstract from above: "The enzyme elastase is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa. Previous studies have shown that expression of the P. aeruginosa elastase gene (lasB) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI). In this study, we analyzed the lasB promoter region to learn more about lasB activation by LasR and PAI. We report that the lasB transcriptional start is located 141 nucleotides upstream from the lasB translational start. It was also discovered that the lasB promoter region contains two putative operator sequences (OP1 and OP2) that are similar to each other and the Vibrio fischeri lux operator. OP1 is located directly upstream from, and may overlap with, the lasB promoter region, and OP2 is centered 102 nucleotides upstream from the lasB transcriptional start site. To study the effects of these putative operators and other sequences upstream from the lasB transcriptional start site on lasB activation, a series of transcriptional lasBp-lacZ gene fusions was constructed. Data from these fusions indicate that both putative operators are involved in LasR- and PAI-mediated lasB activation, with OP1 being more important than OP2."

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 58
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution by iGEM UZurich 2023 Lactoback

Group: UZurich 2022

Summary: Confirmation that RhlR and RhlI inducible to induce the LasB promoter

Contribution

Traditionally, the rhl quorum sensing system uses the RhlAB promotor. However, this part has already been characterised in iGEM. We wanted to increase the iGEM parts collection by confirming that RhlR is also able to induce the LasB promoter (1). 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. However, the strength of the promoters needs to be compared first.

Characterization

Figure 1: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. (BBa_K4662042). 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.

Usage and Biology

The CFP moiety can be easily switch to the sequence of the protein of interest.

Remark

Further experiments were performed which show the auto-induction of the LasB promoter, confirmed with a co-transformation wiht RhlI plasmid. partinfo>BBa_K4662042</partinfo>

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.

Functional Parameters: Austin_UTexas

BBa_R0079 parameters

Burden Imposed by this Part:

Burden Value: 1.2 ± 7.7%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

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

Kumar, L.; Patel, S.K.S.; Kharga, K.; Kumar, R.; Kumar, P.; Pandohee, J.; Kulshresha, S.; Harjai, K.; Chhibber, S. Molecular Mechanisms and Applications of N-Acyl Homoserine Lactone-Mediated Quorum Sensing in Bacteria. Molecules 2022, 27, 7584. https://doi.org/10.3390/molecules27217584).