Team:Wageningen UR/Wetlab/QuorumSensing
One of the pillars on which Cattlelyst is build is biosafety. To ensure biocontainment, three safety mechanisms were designed, of which one is the proximity-based kill switch. Quorum sensing is used to ‘sense’ the cell density. If a bacterium escapes from the biofilter, the low cell density activates the production of a toxin and repress antitoxin production, thereby killing the cell. Using a proof of concept study in which the envisioned toxin is replaced by GFP and the corresponding antitoxin by RFP, the dynamics of the circuit were tested. This showed that medium rich in the quorum sensing molecule, AHL, represses GFP production and activates RFP production. Our biofilter will consist of a co-culture of P. putida and E. coli. We built the circuit in P. putida, and to make E. coli dependent on P. putida, we built the same circuit in E. coli without the ability to produce AHL. A co-culture experiment of P. putida and E. coli showed that P. putida is able to activate the quorum sensing circuit in E. coli. Combined with the observed high AHL sensitivity, we showed that quorum sensing might be a good mechanism to base this safety circuit on.
Introduction
Because we designed our biofilter Cattlelyst for real-world application on cattle farms, biocontainment of the GMO’s is crucial. Therefore, we have designed three layers of safety. The first layer is based on the methane concentration, which is relatively high inside the biofilter, resulting in a methane-dependent kill switch . The second layer is the proximity-dependent kill switch, which is linked to the methane-dependent kill switch, as proximity is an input signal for the hybrid promoter that control toxin production in that kill switch. Lastly, the third layer is based on auxotrophy, which makes Escherichia coli and Pseudomonas putida dependent on each other, making them unable to escape from the biofilter by themselves. Combining these three layers of safety ensures total dependency of the two bacterial species with the biofilter conditions and with each other, making them unable to escape from the biofilter.
Quorum sensing
Quorum sensing is the ability of microorganisms to 'sense' the cell density by exchanging signalling molecules [1]. The signalling molecules can diffuse out of the cell when they are produced. When the cell concentration is high enough, the concentration of the signalling molecules increases, activating or repressing repress the quorum sensing-related promoters. In Vibrio fischeri, the quorum-sensing molecule acyl homoserine lactone (AHL) is produced by the LuxI (BBa_C0061) protein. AHL binds to LuxR, which can either activate the lux pR promoter (BBa_R0062) or repress the lux pL promoter (BBa_R0063) [2], [3].
Toxin-antitoxin system: hok/sok
To link the cell density to cell survival, this quorum sensing circuit needs to be coupled to something that can kill the cell. For this, we chose the hok/sok toxin-antitoxin system (BBa_K1783001). In its natural context, the system ensures plasmid maintenance [4], [5]. The Hok protein can kill the cell by damaging the cell membrane [4], [6]. Translation of the hok mRNA is inhibited by sok RNA by binding the hok mRNA, thereby blocking ribosome access [6]. When the cell loses the plasmid, there is no new transcription of hok and sok (m)RNA. The sok RNA is very unstable, with a half-life around 30 seconds, while the hok mRNA is relatively stable with a half-life of 20 minutes [7]. After plasmid loss, only hok mRNA will be left in the cell which is translated to Hok protein, killing the cell [4], [8].
Safety circuit
The quorum sensing and hok/sok toxin-antitoxin system will be coupled as follows; The hok gene is put under the control of the lux pL promoter, which is inhibited by the AHL-LuxR complex. The sok gene is put under the control of the lux pR promoter, which is activated by the AHL-LuxR complex at high cell densities (see Figure 1). When the cells are inside the biofilter, where the cell density is high, the toxin production is inhibited while the antitoxin production is activated (see Figure 2). When a cell escapes from the biofilter, the cell density is low, so the toxin production is no longer inhibited while the antitoxin production is not activated anymore (see Figure 3). The hok mRNA accululates, so the toxin protein is produced, leading to cell death. This circuit is build in P. putida. In our biofilter, we have a co-culture of E. coli and P. putida. We built a similar system in E. coli, without the ability to produce AHL. This adds an extra layer of safety (see Figure 4), as E. coli is now dependent on P. putida for AHL, while P. putida is also dependent on E. coli because of the carbon source-dependency, making them unable to escape alone.
To test this system, a proof of concept study is performed in which hok and sok are replaced by a fluorescent reporter. This is done to prevent cloning difficulties and because fluorescent proteins are easy to measure. The goal is to make P. putida activate the quorum sensing circuit in E. coli. To achieve this the following questions need to be answered:
- Is the lux pR promoter activated by AHL rich medium?
- Is the lux pL promoter downregulated by AHL rich medium?
- Are the lux pR and lux pL still activated and repressed respectively when present in one strain?
- In a co-culture of E. coli and P. putida, can P. putida activate the lux pR promoter of E. coli?
Approach
The circuit was tested by a proof of concept in which the hok and sok genes are replaced by GFP and RFP. The goal of this proof of concept is to test whether the quorum sensing part of the circuit works and if P. putida is able to activate the quorum sensing circuit in E. coli. The construct that are designed to test this are explained below.
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Designed constructs
arrow_downwardThe constructs shown here are build in the plasmid backbones SEVA64 and SEVA23. Wild type strains are strains that only have the plasmid backbone without an insert.
Figure 5: GFP is under control of the lux pR promoter which is activated by the luxR-AHL complex. LuxR is constitutively expressed. In this construct, no AHL is produced, so the lux pR promoter should only be induced when AHL is present in the medium. 
Figure 6: This construct is the same as in Figure 5 except for that LuxI is also produced under control of the lux pR promoter. Here the lux pR promoter should be activated in high cell densities without addition of AHL in the medium. 
Figure 7: This contruct is almost the same in Figure 5. Here GFP is replaced by RFP to be able to combine contructs in a co-culture or in one strain. 
Figure 8: This construct is the same as in Figure 6, but GFP is replaced by RFP. 
Figure 9: Here GFP is under control of the lux pL promoter which is downregulated by the LuxR-AHL complex. As no AHL is produced by this complex, the promoter is active when there is no AHL in the medium. Where there is AHL in the medium, it should be downregulated. 
Figure 10: Like in the construct of Figure 9, GFP is under control of the lux pL promoter, but here luxI is produced under control of the lux pR promoter. Therefore, in high cell densities, the production of GFP should be downregulated, also when no AHL is added to the medium.
The constructs were transformed in E. coli K12 (MG1655). To test if the designed constructs were activated by AHL, plate reader experiments were performed in which both the OD and the fluorescence were measured over time.
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Plate reader experiments
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- Day 2
- Day 3
Make a pre-culture of the positive control strain in LB.
Make pre-cultures of the positive control, reporter and wild type strain in LB.
Wash the pre-culture of day 1 in the media that will be used for the plate reader experiment and make a new pre-culture in that medium. As initially M9 media gave growth issues, we used 80% M9 (supplemented with 0.2% glucose) and 20% LB.
Spin off the pre-culture of the posivite control in 80% M9 medium and filtersterilze the supernatant. Spike the medium with 0.2% glucose. This conditioned medium (CM) is rich in AHL. Dilutions of the CM medium were made by adding fresh 80% M9 + 20% LB medium.
Wash the LB pre-cultures of the positive control, reporter and wild type strain in 80% M9 + 20% LB medium.
A 96 wells plate was prepared with the three strains (inoculated with OD=0.5) in triplicates and with different dilutions of the CM. Blanks were used to correct the OD for background absorption. The fluorescence of the positive control and reporter strain was corrected by substracting the fluorescence of the wildtype strain.
As both the lux pL and lux pR promoter need to work simultaneously, E. coli K12 was transformed with both the reporter-pR-RFP and reporter-pL-GFP construct. With this strain, plate reader experiments were performed.
To test if the quorum sensing circuit in the reporter E. coli strain can be activated in co-culture conditions, an experiment was conducted where two E. coli strains were grown together on a agar plate. The two strains used were the AHL-producing positive control-pR-RFP strain and the reporter-pR-GFP strain. Different dilutions of an overnight (ON) culture of the positive control and wild type strain were plated. On top of this, drops of different dilutions of the reporter strain were added and the plates were incubated overnight. The next day, pictures of the fluorescence of the plates were made. This experiment was repeated with the co-culture of P. putida and E. coli.
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Protocol co-culture experiment on agar plates
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- Day 2
- Day 3
ON cultures of the WT E. coli and P. putida strains, Reporter-pR-GFP E. coli strain (Figure 5) and positive control-pR-RFP WT E. coli and P. putida strains (Figure 8) were made.
All cultures were diluted to OD = 0.5.
Five dilutions were made for all cultures ranging from 101-105.
From each dilution, 200µL of either the WT or positive control strain was spread on a agar plate with LB+agar.
To each plate, a drop from each dilution of the reporter-pR-GFP strain was added.
Plates were incubated overnight.
Pictures of the GFP fluorescence were made.
Results
To investigate the dynamics of the designed constructs explained in Figures 5-10, plate reader experiments were done. The fluorescence was divided by the optical density and plotted over time (see Figure 14 – 16). Bargraphs were made at t=11 hours (see Figures 11 – 13). The bargraphs and time series plots show an increase in fluorescence of the reporter strain with the lux pR promoter when AHL is present in the medium compared to when no AHL is present in the medium (see Figures 11 – 12). For the constructs in which have the lux pL promoter, the time series plots and the bargraphs show that AHL in the medium downregulates GFP production (see Figure 13).
Separate constructs
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Time series plots
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Figure 14: Time series plots of the reporter-pR-GFP and positive control-pR-GFP strain grown in different concentrations of CM rich in AHL. Fluorescence was corrected by the OD.
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Time series plots
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Figure 15: Time series plots of the reporter-pR-RFP and positive control-pR-RFP strain grown in different concentrations of CM rich in AHL. Fluorescence was corrected by the OD.
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Time series plots
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Figure 16: Time series plots of the reporter-pL-GFP and positive control-pL-GFP strain grown in different concentrations of CM rich in AHL. Fluorescence was corrected by the OD.
Combined construct
As both the lux pL as the lux pR need to work simultaneously in E. coli in our final circuit, both the reporter-pR-RFP and reporter-pL-GFP constructs were put in one E. coli strain. The plate reader experiment was repeated with this strain and showed that the constructs also show the desired dynamics when present simultaneously (see Figure 17).
For the functioning of the biosafety circuit, the toxin/antitoxin ratio in high cell densities versus low cell densities is important [9]. To obtain a sufficient big change in the toxin/antitoxin ratio in high versus low cell censities, the circuit needs to be sensitive to an input signal, in this case AHL concentration. The sensitivity of the circuit to the AHL concentration (with is related to the cell density) is calculated by;
As can be seen in Figure 18, the circuit is sensitive to the cell density and the sensitivity increases over time.
AHL medium P. putida
The AHL in the biofilter will be produced by P. putida, therefore a plate reader experiment was conducted where the AHL in the CM was produced by P. putida instead of E. coli. This experiment showed that CM produced by P. putida can activate the quorum-sensing promoter in E. coli (see Figure 19).
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Time series plots
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Figure 20: Time series plots of the reporter-pR-GFP strain grown in different concentrations of CM rich in AHL produced by P. putida. Fluorescence was corrected by the OD.
Co-cultures
As the CM produced by both E. coli and P. putida showed activation of the lux pR promoter, an experiment was done to investigate if this activation is also obtained in co-culture conditions. The positive control-pR-RFP strain of E. coli and P. putida were grown on an agar plate with the reporter-pR-GFP E. coli strain. Pictures of the fluorescence of the plates showed activation of the reporter-pR-GFP E. coli strain when grown together with AHL producing E. coli or P. putida strains.
Conclusion
To test the dynamics of the quorum sensing part of the safety circuit, a proof of concept study is performed in which hok (toxin) is replaced with GFP and sok (antitoxin) by RFP. The quorum sensing constructs with the lux pR promoter were shown to be activated by the conditioned medium rich in AHL produced by both E. coli and P. putida and the lux pL promoter was repressed by the conditioned medium. Both promoters were still activated or repressed when they were both present in the same strain, and were showing a high sensitivity to AHL. A co-culture experiment done on agar plates showed that in co-culture conditions where P. putida is producing AHL, the quorum sensing circuit with the lux pR promoter in E. coli is activated. These results show that quorum sensing is a suitable input signal for the proximity-dependent kill switch. Replacing the fluorescent proteins with the envisioned toxin and corresponding antitoxin, could lead to a safety circuit that is able to kill any escaper cell.
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References
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