Partnership

Contents


Background

Fenitrothion Biosensor

3-PBH Kill Switch

Background

With our matching aspirations for clearing pesticide build-up in the environment, we partnered with CyanoClean, the 2022 Concordia University iGEM team who are working on organophosphate pesticide degradation. We adopted our aptamer-based biosensor to one of CyanoClean’s target pesticides — fenitrothion, while they designed a toxin/anti-toxin biocontainment strategy for our engineered E. coli in return.

We held a meeting in early July with the Concordia iGEM team, CyanoClean, due to the similarities between our projects. We both wanted to utilise synthetic biology to tackle the problem of pesticide remediation within the environment. From our early discussions, it was evident that both our teams shared similar concerns regarding biocontainment, as both our projects involve the proposed release of our genetically engineered organisms (Pyre: E. coli; CyanoClean: cyanobacteria) into the natural environment. At this point, we gained interest for CyanoClean’s preliminary plans to integrate a nitrophenol dose-dependent kill switch, whilst they gained interest for our plans in developing a cell-free aptamer-based biosensor.

Subsequently, a partnership between both our teams was proposed; CyanoClean would develop a kill switch that is suitable for our engineered E. coli, whilst Pyre would develop a sensing system suitable for their target organophosphate pesticide. Subsequently, weekly meetings were established for progress updates and exchanging advice. This partnership was deemed to be immensely successful, as it enabled both our teams to incorporate vital sensing and biocontainment components into our projects, alongside our initial degradation plans. Hence allowing the production of a complete flowing system, for the remediation of our target pesticide residue. This would not have been possible without the establishment of this essential symbiotic partnership.

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Figure 1. Collage of screenshots from our weekly meetings.

Development of Aptamer-Based Biosensor for Fenitrothion (Pyre)

In order to fulfil our obligations to CyanoClean, we developed of a sensing system for CyanoClean’s target pesticide fenitrothion by adapting our optimised protocol for the creation of our aptamer-based biosensor for λ-cyhalothrin. Subsequently, we plugged-in 2 aptamers (FenA1 and FenA2) targeting fenitrothion into our biosensor system, with tests running in-parallel. These aptamer sequences were derived following a thorough literature review (Parts Overview). These sensing sets were run using 40 μL PDDA, 10 μL 500 mM MOPS buffer, 15 μL aptamer (FenA1/FenA2), 30 μL of 10mg/mL fenitrothion pesticide, and 60 μL AuNP, followed with a 10 minute incubation at 30°C (See Design and results for information regarding function of biosensor).

In our detection assay to test whether the two adapted systems are able to indicate the presence of fenitrothion, a significant colour change to blue was recorded in samples containing the pesticide, while controls (without pesticides) gave off a red hue (Figure 2). It is evident that the aggregation of AuNP was due to the presence of fenitrothion as it does not occur in the absence of this pesticide.

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Figure 2. Aptamer-based biosensing system inducing a red to blue colour change in the presence of 10 mg/mL fenitrothion pesticide. +ve: positive control (total AuNP aggregation); -ve(1): negative control (no AuNP aggregation) with FenA1 aptamer; -ve(2): negative control (no AuNP aggregation) with FenA2 aptamer; FenA1: sensing system with pesticide and FenA1 aptamer; FenA2: sensing system with pesticide and FenA2 aptamer.

Using a spectrophotometer, these observations were quantified as absorbance values, with readings taken at 528 nm (red) wavelength and 650 nm (blue) wavelength. These were then used to calculate absorbance ratios (650 nm/528 nm), with higher values indicating the increased aggregation of AuNP, and a greater intense blue intensity. Subsequently, we observed that in the presence of 30 µL 10mg/mL fenitrothion, both FenA1 and FenA2 aptamer systems had an absorbance ratio very close to the control without aptamers (i.e. AuNP fully aggregated form), at ~0.8. In comparison in the absence of the pesticide, the values were noticeably lower, at ~0.3. These quantitative results correspond to the visual colour changes observed, whilst indicating the capacity for adapting the aptamer-based biosensing system from our λ-cyhalothrin target to CyanoClean’s fenitrothion.



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Figure 3. Absorbance ratios calculated for the sensing tests ran with 10 mg/mL fenitrothion with either FenA1 and FenA2 aptamer; aggregation occurs with both aptamers when in the presence of the pesticide, indicating the success of the aptamer-based biosensor. No aptamer: Positive control (total AuNP aggregation); FenA1: Sensing system with FenA1 aptamer and 10 mg/mL fenitrothion; FenA1 + no pesticide: Sensing system with FenA1 aptamer and no pesticide; FenA2: Sensing system with FenA2 aptamer and 10 mg/mL fenitrothion; FenA2 + no pesticide: Sensing system with FenA2 aptamer and no pesticide.

Once we confirmed that the system was able to sense the presence of fenitrothion, we went on to test whether the degree of colour change is concentration dependent. 30 µL fenitrothion of varying concentrations (1mg/mL to 5mg/mL, in 1mg/mL intervals) were made up from the stock 10mg/mL, via dilution in acetone; FenA2 was chosen as the aptamer of choice for running these sensing tests, as it was observed to be the most sensitive aptamer. FenA2 had a higher absorbance ratio of ~0.85 compared to that of ~0.76 for FenA1 (Figure. 3). A gradient from red to blue colour change was seen with the increase in fenitrothion concentration, which is consistent with that of our own pesticide target λ-cyhalothrin. Closer quantitative analysis of the absorbance ratios were conducted, which saw a gradual increase from ~0.30 at 0mg/mL of fenitrothion, to ~0.75 at 5mg/mL (Figure. 4). Therefore, this provided evidence for the adoption of the aptamer-based biosensor for fenitrothion, with both visual and quantitative results describing the red to blue colour change in the presence of that specific pesticide.

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Figure 4. Varying intensities of red to blue colour change, dependent on the concentration of fenitrothion present within the aptamer-based biosensor. +ve: positive control (total AuNP aggregation); -ve: negative control (no AuNP aggregation); 1: 1 mg/mL fenitrothion; 2: 2 mg/mL fenitrothion; 3: 3 mg/mL fenitrothion; 4: 4 mg/mL fenitrothion; 5: mg/mL fenitrothion; 10: 10 mg/mL fenitrothion.
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Figure 5. Absorbance ratios calculated for the sensing tests ran with varying concentrations of fenitrothion (1mg/mL to 5mg/mL in 1mg/mL intervals). Increasing AuNP aggregation is dependent on the increasing concentrations of fenitrothion within the sensing system, further contributing to increasing intensities of red to blue colour change.

Finally, to test the specificity of the fenitrothion aptamer, 5mg/mL λ-cyhalothrin was added to the most sensitive fenitrothion and λ-cyhalothrin systems. The absorbance ratio of the fenitrothion sensor with λ-cyhalothrin remains at the level before the addition of the pesticide, which was roughly half of that of the control with no aptamers (i.e. AuNP fully aggregated). In contrast, there was a significant rise in the absorbance ratio in that of the λ-cyhalothrin system to a level comparable to the control. These indicate that the fenitrothion aptamer sensor has low specificity to non-targeted pesticide such as λ-cyhalothrin, which further supports the capacity to adapt our aptamer-based biosensor to support fenitrothion.



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Figure 6. Aptamer-based biosensors for both λ-cyhalothrin (with LCT-3 aptamer) and fenitrothion (with FenA2 aptamer) running in-parallel to potentially detect the presence of 5 mg/mL λ-cyhalothrin. No aptamer: Positive control (total AuNP aggregation); LCT-3: Sensing system with LCT-3 aptamer and 5 mg/mL λ-cyhalothrin; LCT-3 + no pesticide: Sensing system with LCT-3 aptamer and no pesticide (negative control); FenA2: Sensing system with FenA2 aptamer and 5 mg/mL λ-cyhalothrin; FenA2 + no pesticide: Sensing system with FenA2 aptamer and no pesticide.

Development of a Kill Switch for 3-PBH (CyanoClean)

It was incredibly vital that our project covers biocontainment strategies for our engineered E. coli, as our project relies upon the proposed release of our genetically modified organism into the environment. Discussions with CyanoClean regarding the nature of our E. coli chassis, enabled them to design a kill switch that would work for our project.

As our E. coli chassis has been genetically modified to express a carboxylesterase enzyme via cell-surface expression, both CyanoClean and Pyre thought it would be best to design a kill switch in which its activation is also induced on the cell surface. Multiple different kill switch approaches were suggested including a pH dependent kill switch, an inducible kill switch which deactivates the E. coli in the absence of the pesticide, and a kill switch which is dependent on a specific protein that is cleaved inside the cell, in which cleavage is promoted via an extracellular stimulus (i.e., a specific metabolite that is not commonly found in the environment). However, many of these kill switch proposals had various limitations, most of which were a result of the unpredictable nature of the environment, resulting in them being unsuitable for our project.

Following conversations with one of our key stakeholders, Syngenta, we learnt that λ-cyhalothrin is naturally able to diffuse across the cell membrane of E. coli, and enter the cell. We passed this new vital information to CyanoClean, who utilised this newfound knowledge to develop a EcoRIR-EcoRIM kill switch (EcoRI restriction-modification system à la toxin/antitoxin kill switch), which is activated by 3-phenoxybenzoic acid (3-PBA) – the first intermediate product along the degradation pathway of λ-cyhalothrin. For this kill switch to work, it relied upon a baseline expression of a toxin which would normally be inhibited by an anti-toxin when in the presence of λ-cyhalothrin. The engineered E. coli would continue to degrade the residues present. However, once the pesticide has been broken down and the concentration of 3-PBA in the environment rises, the kill switch becomes activated. Anti-toxin expression is inhibited, and is therefore no longer able to suppress the toxin effects. Thus, toxin concentration rises, and upon reaching an optimal concentration, it will effectively kill the E. coli chassis; a biocontainment strategy for our genetically modified organism has been successfully integrated into our project.

To understand more about the technicalities behind the implementation of the our kill switch designed by CyanoClean, head over to the Concordia iGEM 2022 partnership page! (https://2022.igem.wiki/concordia-montreal/partnership)