Team:NJMU-China/Results

Overview

As for the lab work, we have completed the design and synthesis of plasmids as well as the optimization of the color-reaction reporting system. However, held by the pandemic of COVID-19, we didn’t have access to lab works until August. Foreseen this situation, we have initiated our modeling project to simulate the whole interaction as a preliminary estimation of part function (See More in Our MODEL).

Design of Serotonin Biosensors

There exist no designed biosensors against serotonin. Therefore, we decided to design the serotonin biosensor from scratch. Through massive literature investigation, we found that in a novel mouse infection model 1, serotonin, acting as an interboundary signaling molecule through quorum sensing, stimulates the production of bacterial virulence factors and increases the formation of biofilms. Specifically, no response was noted in the rhl cellular system, indicating specificity for the las QS pathway.

Based on this knowledge, we referenced the design of biosensors aimed to detect QS molecules 2. The biosensor plasmid is based on a typical heterologous expression system, pGEX-6p-1 vector and contains two parts. The first part (Sensor-1) expresses LasR molecule under the control of Ptac promoter and lac operator (activated in the presence of IPTG). The second part (Sensor-2 & Reporter) is under the control of the inducible promoter PlasI and expresses beta-galactosidase, which is a classic reporter gene. In the presence of serotonin or Quorum-Sensing molecules (e.g. HSL or AHL), they will bind with LasR to form a transcription activating complex targeting PlasI. We aimed to visualize the result through color-reactions by beta-galactosidase and X-gal.

Synthesis and Modification of Plasmids

1. Synthesis of Sensor-1

The gene sequences corresponding to lasR from the lasR/lasI AHL-mediated QS regulatory system of Pseudomonas aeruginosa were synthesized using polymerase chain reaction (PCR) (https://www.ncbi.nlm.nih.gov/gene/881789). The plasmid pGEX-6p-1 was prepared by inserting a 5' BamHI/EcoRI, 3' EcoRI fragment containing the lasR sequences at the downstream of GST and HIV 3C site (Figure 1). The resulting pGEX-6p-1 vector (Serotonin Biosensor Version 0) contains the lasR gene which is under the transcriptional control of Ptac.

Figure 1. Selection of Restriction Endonuclease Sites

The construction of Serotonin Biosensor Version 0 was verified by digesting the plasmid with BamHI and EcoRI and confirming the lengths of the digested fragments on a 1% agarose gel in 37°C water-bath for 40 minutes (Figure 2). We have also sequenced the engineered part to ensure there are no mutations.

Figure 2. Enzyme Digestion Result of Serotonin Biosensor Version 0

2. Synthesis of Sensor-2 & Reporter

The gene sequences corresponding to PlasI were obtained from the paper 'The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter' published in J Bacteriol 3 (Figure 3). Sequences of related protein ORF were acquired from NCBI: https://www.ncbi.nlm.nih.gov/gene/881777. We further used homologous recombination methods to integrate this DNA fragment into Serotonin Biosensor Version 0. The resulting plasmid (Serotonin Biosensor Version 1) contains the lacZ gene which is under the transcriptional control of PlasI.

The construction of Serotonin Biosensor Version 1 was verified by digesting the plasmid with Clal and Xhol and confirming the lengths of the digested fragments on a 1% agarose gel in 37°C water-bath for 40 minutes (Figure 4). We have also sequenced the engineered part to ensure there are no mutations.

Figure 4. Enzyme Digestion Result of Serotonin Biosensor Version 1

3. Optimization of the color-reaction reporting system

Before characterization of our constructed parts, we have optimized the color-reaction reporting system as we have introduced other reagents like standard serotonin in the system. Through gradient-condition experiments using wild-type pGEX-6p-1 vectors，we have found the optimal amount of IPTG and X-gal as shown below:

4. Characterization of Serotonin Biosensor Version 1

The plasmid Serotonin Biosensor Version 1 was transformed into competent E. coli DH5-Alpha cells using the standard protocol provided by the manufacturer 4. The transformed cells were grown at 37 °C overnight on LB agar plates containing 100 µg/mL ampicillin. Cells from a single colony were grown overnight at 37 °C, with shaking at 250 rpm in LB broth containing the same amount (w/v) of ampicillin. Finally, glycerol stocks were prepared from these cell cultures and stored at -80 °C. Fresh cell cultures were obtained from the glycerol stocks and grown at 37 °C, with shaking at 250 rpm in LB broth, containing 100 µg/mL ampicillin, until the desired optical density at 600 nm (OD 600 nm) was reached.

Further, we characterized the function of Serotonin Biosensor Version 1 by standard serotonin and N-3-oxo-dodecanoyl homoserine lactone (positive control). However, we kept achieve negative results in our experiments. Therefore, we went through some trouble-shooting experiments as in the scheme below:

After incubation with IPTG, we extracted whole protein of cells and ran an SDS-PAGE test (Figure 5). Results indicate that, there may exist problems with low transcriptional activity of the inducible promoter PlasI.

Figure 5. SDS-PAGE Result of Extracted Proteins after IPTG Induction

4. Modification to Improve Promoter Activity of PlasI: Serotonin Biosensor Version 2 & 3

We have turned to many ways to improve the transcriptional activity of PlasI, including searching the part registry run by the iGEM community. Finally, we were enlightened by the natural function of lasI gene, which encodes acyl-HSL synthase enzymes (LasI) that is responsible for the synthesis of the N-3-oxo-dodecanoyl homoserine lactone (3-O-C12-HSL) 5. 3-O-C12-HSL, Quorum-Sensing molecule of P. aeruginosa, activates the LasR/LasI signaling pathway in turn.

Based on this whole circle of positive feedback, we modified the plasmid Serotonin Biosensor Version 1 by expressing a fusion protein of LasI and beta-galactosidase.

The construction of Serotonin Biosensor Version 2 was verified by digesting the plasmid with EcoRI and confirming the lengths of the digested fragments on a 1% agarose gel in 37°C water-bath for 40 minutes (Figure 6).

Figure 6. Enzyme Digestion Result of Serotonin Biosensor Version 2

However, as we routinely sequenced the engineered part, we found a frame-shift mutation brought by mis-designing of sequence. Therefore, we re-constructed Serotonin Biosensor Version 3 under the same scheme. We expect further characterization can be accomplished in this November.

References

1 Knecht, L. D. et al. Serotonin Activates Bacterial Quorum Sensing and Enhances the Virulence of Pseudomonas aeruginosa in the Host. EBioMedicine 9, 161-169, doi:10.1016/j.ebiom.2016.05.037 (2016).
2 Saeidi, N. et al. Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol Syst Biol 7, 521, doi:10.1038/msb.2011.55 (2011).
3 Rampioni, G. et al. The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter. J Bacteriol 188, 815-819, doi:10.1128/JB.188.2.815-819.2006 (2006).
4 Struss, A., Pasini, P., Ensor, C. M., Raut, N. & Daunert, S. Paper strip whole cell biosensors: a portable test for the semiquantitative detection of bacterial quorum signaling molecules. Anal Chem 82, 4457-4463, doi:10.1021/ac100231a (2010).
5 Jayaprada, T. et al. The interference of nonylphenol with bacterial cell-to-cell communication. Environ Pollut 257, 113352, doi:10.1016/j.envpol.2019.113352 (2020).