Vision

For our prototype, we envision a convenient spray bottle that contains a solution of our purified colored residue-binding proteins. Users will apply the solution to the surface of the produce, and if there are contaminants, the proteins will attach and appear as a patch of color. We attached a His Tag to our binding proteins so they can be purified if desired by the consumers. Alternatively, proteins can be kept inside bacterial cells, so there are two ways in which our product can be used to detect agricultural contaminants.

Metal-binding Prototype

Initially, we created a protocol to test our construct Met + RFP, which binds to nickel ions, and BacFerr + RFP, which binds to iron ions, in realistic conditions since these constructs showed the optimal binding results from our Experiments. For more information on binding data, refer to our Experiments page.

For our prototype, we first grew liquid cultures of Met + RFP, BacFerr + RFP, and RFP only (our control). Once we confirmed that they were all visibly red, we prepared pieces of onion that will represent the produce. Then, we made metal ion solutions of 25mM NiSO₄ and 15mM FeSO₄ and applied the solutions and water (a control) onto the pieces of onion to simulate heavy metal contamination of produce. From our previous experience, we noticed that when we left the metal ion solution on the produce for too long, the produce turned brown. This not only gave us a greater incentive to solve the issue of metal contamination but also informed us that we needed to quickly dry the pieces of onion to prevent the browning for our experiment. We applied different metal ion solutions onto pieces of onion, which were dried quickly. We then applied each of the metal binding proteins to its respective ion-soaked onions and waited for different time intervals before removing the solutions from the onion. To indicate the presence of heavy metals, we looked to see if the experimental sets were more red than the control sets.

Figure 1.
Experimental Set-up for Metal-Binding Prototype.

However, we were unable to conclusively decide visually whether the proteins were bound to the heavy metal ions or if they were just laying on top of the surface of the onion. Even if the red proteins were bound to the metal ions, it is possible that the concentration could be too low for visualization. Thus, we turned to modeling to help us understand the binding interaction of our proteins and agricultural residues.

Our modeling allows us to find the maximum metal ion concentration we can detect at a certain cell concentration. The model works on the assumption that if we see color on a contaminated surface, then the colored proteins are all bound to the heavy metal ions. With this relationship, we can determine the metal ion concentration that the proteins are bound to, and also the concentration of cells needed to detect the visible threshold of heavy metal ion concentration, which is 2.876×10¹⁸ cells/cm². Since we were unable to see a patch of color remaining on our onions, it is possible that the heavy metal ion concentration was below the visible threshold, and thus the concentration of cells we used is lower than what is needed to detect the threshold. To find out more about how we determined the concentration of cells needed to detect the visible threshold of heavy metal ions, refer to our Modeling page.

Pesticide-binding Prototype

Similar to our metal-binding prototype, we envision a spray that will interact with pesticides to provide visual detection. We designed two constructs that produced enzymes OpdA or LC cutinase, which interact with organophosphate pesticides, and attached a blue chromoprotein amilCP. We chose malathion (mal) as our model pesticide to test because it is a commonly used organophosphate. LC cutinase degrades malathion into monoacid and diacid, and OpdA cleaves malathion to expose its sulfhydryl group (Scott et al., 1970). While these constructs showed color on agar plates, and their sequences were confirmed to be correct, they surprisingly did not exhibit color in liquid cultures. Nevertheless, we still wanted to demonstrate our prototype with these constructs.

We realized we could detect the products of malathion degradation to show how our prototype would work. Our best results came from when we utilized the reaction of the OpdA enzyme with malathion, which produced sulfhydryl groups that can be detected by Ellman’s reagent to produce a visible yellow color (ThermoFisher, 2011). We modified the Ellman’s test protocol from our Experiments page to show how our prototype will work in realistic conditions. We expected that since the OpdA protein breaks down malathion and exposes its sulfhydryl groups, we could add Ellman’s reagent to a mixture of OpdA and malathion for visual detection. You can refer to Experiments page for more information on the Ellman’s test.

We cut out pieces of parafilm, which represented the surface of a produce, and applied either a drop of distilled water, as a control, or a drop of malathion that was at a concentration recommended by its producer. Then, we applied purified proteins of OpdA or OpdA + amilCP onto the surface, which mimics how users would spray their produce with the solution containing our proteins.

Before adding Ellman’s reagent:

Figure 2. Before adding Ellman’s reagent.
We added 225 µL of either OpdA or OpdA+amilCP to 150 µL of either H₂O or
malathion (mal). Prior to adding Ellman's reagent, all solutions were clear.

Finally, we added Ellman’s reagent to the mixture. We expected that the mixture containing OpdA or OpdA + amilCP and malathion would turn yellow once Ellman’s reagent was added.

We completed a visual test of the control group and the group with malathion, since users would also have to rely on visual comparison. We looked for a visible difference in yellow between the control group and malathion group.

After adding Ellman’s reagent:

Figure 3. After adding Ellman’s reagent.
Upon addition of Ellman's reagent, we observed more yellow in the samples where
malathion (mal) was added to OpdA or OpdA+amilCP compared to those where
only water was added. The results suggest that our purified OpdA proteins
successfully cleaved malathion to reveal thiol groups, which Ellman’s reacted with.

Here is another look at the prototype:

Figure 4.
After adding Ellman’s reagent.This is the same as the previous figure, except in a
different configuration and a clearer comparison between the experimental sets and
control sets.

Figure 5. We quantitatively measured the absorbance change at 412.4 nm when our
purified construct with OpdA was treated with malathion vs only water as a control.

This experiment shows that our proteins reacted with the malathion present, as Ellman’s reagent detected the exposed sulfhydryl groups on cleaved malathion and turned yellow immediately. From this, we can conclude that our prototype works in realistic conditions as it does not require advanced lab equipment to visualize.

Although this prototype differs slightly from our original vision of colored residue-binding proteins, this prototype still gives consumers an easy, quick, and convenient method to detect residues on their produce.

References

DTNB (Ellman's Reagent) (5,5-dithio-bis-(2-nitrobenzoic acid). (2011). Retrieved October 19, 2019, from https://www.thermofisher.com/order/catalog/product/22582.