We have engineered a biosensor based on LasI-LasR quorum sensing circuit for the detection of P. aeruginosa-specific quorum sensing molecules, N-3-oxo-decanoyl L-homoserine lactone (C10HSL) and N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) as low as 1x10-11M. In addition, the engineered biosensor had high sensitivity in response to the quorum sensing molecules, 3OC12-HSL with an EC50 value of 2.823 x 10-11M. In the process of biosensors construction, we faced many challenges in an attempt to engineer a sensitive biosensor with high green fluorescence readout. We believed that our modifications that we made in the protocol and the new composite part (BBa_K4946004) that we constructed provide valuable insights for future iGEM team to design whole-cell biosensors implemented with genetic circuits.
Modifications
1. Construction of biosensors using different pET vectors and inducible promoters
Our biosensor has two modules: LasR sensing module and EGFP reporting module. The transcription factor LasR was expressed under the control of the constitutive T7 promoter in the plasmid pET 21b and pET 23b. Two pET plasmids were used in order to compare if the presence of lac operator in pET 21a can prevent leaky expression of the LasR and thus give less background fluorescence signals in the uninduced state. Besides, we used two LasR inducible promoters, pLasRL and pLasR3 in the reporting module in order to compare the two promoter strengths. The two modules were transformed into DH5α individually. The extracted DNA of the two modules were then co-transformed into the BL21 C41(DE3)pLysS.
However, our results showed that there was no significant difference in EGFP production among the four biosensors indicating that the uses of either pET 21b and pET 23b and either pLasRL and pLasR3 in the expression plasmid exhibit no differences in the functionality of engineered biosensors.
2. Construction of biosensors using different ratio of sensing module and reporting module
We hypothesized that the sensing module in the expression plasmids need to be in excess so that more AHL molecules can bind and drive the expression of reporting module. We transformed three different ratios of the LasR sensing module and the EGFP reporting module in pET21b-LasR-pUC57-pLasRL-EGFP construct including 1:1, 2:1 and 3:1 sensing module to reporting module. Our results demonstrated there was no significant difference between the different ratios of the sensing module and the reporting module and EGFP production.
3. Two individual modules in two expression plasmids vs two modules integrate into a single expression plasmid
We find that the biosensors engineered by co-transforming two individual modules into host cells showed moderate fluorescence signals after 15h. However, the strategy of constructing a biosensor by integrating two modules in a single plasmid and then transform into host cells yielded higher EGFP production within a shorter time. We speculate that it is easier for bacteria to take up single plasmids than two different plasmids.
4. Autofluorescence observed in fluorescence microscope
In our experiment, we used various concentrations (1x10-3M to 1x10-11M) of synthetic AHL molecules to investigate the functionality of biosensor. We observed high autofluorescence emitted from biosensor cells under fluorescence microscope. Then we reduce the use of DMSO in the preparation of diluted AHL molecules. We carry out serial dilution with MilliQ water instead of DMSO. We found the changes can reduce autofluorescence from biosensor cells. We also found that LB medium has high RFU value and would affect the result when quantifying EGFP production from the construct. Cell washing step is necessary for both fluorescence microscope imaging and EGFP production measurement.
5. Use Fluorescein and J36400 from iGEM measurement kit to calibrate and test the detection limit of microplate reader
We also used a fluorescence chemical named fluorescein. We obtained the optimal concentration by serial dilution and then measured its RFU under the microplate reader in order to calibrate it. Fluorescein can act as a positive control as it shows the most optimal situation where constant fluorescence is emitted. We followed the protocol provided by the iGEM interlaboratory study via the following link:
https://technology.igem.org/interlabs/2023
We also measured fluorescence of Part: J364000 which is a GFP expressing constitutive device. This construct can constantly produce the GFP protein. We transformed the plasmid into BL21 and then quantified the fluorescence of GFP protein using the microplate reader.
It can act as a positive control as it shows the most optimal situation where constant fluorescence is emitted. The part was obtained from iGEM 2023 measurement kit.
Our new composite part (BBa_K4946004) can add new data collected from fluorescence microscope to an existing part BBa_C0179, lasR activator from P. aeruginosa PAO1.
In our project, transcription factor LasR was expressed in the sensing module, which can bind to the synthetic AHL molecules to form LasR-AHL complex. The complex can then bind to inducible promoter, pLasRL to activate the EGFP production. BBa_C0179 was used as the LasR transcription factor in the sensing module of our composite part. Our results showed that in the presence of the 1x10-7M AHL molecules, C10HSL and 3OC12-HSL, the engineered biosensor cells expressing LasR in the sensing module produced strong green fluorescence signals while in the presence of C4HSL and in the control set-up, there was no green fluorescence signals observed. This showed that the LasR sensing module was able to detect C10HSL and 3OC12-HSL.
The results demonstrated that EGFP production rate of the biosensor was the highest when it was incubated with 3OC12-HSL followed by C10HSL. The biosensor did not show response when it was incubated with C4HSL.
To conclude, our data showed that LasR activator from P. aeruginosa PAO1 (BBa_C0179) can detect AHL molecules, 3OC12-HSL and C10HSL.
