The concept for the project was initially inspired by a concept presented during WPI’s Synthetic Biology Capstone course. The project, entitled At-Home pH Based Lead Detection, originally envisioned a proof of concept plasmid design containing a proton pump inserted into a pBRr vector contained by E. coli bacteria. In the presence of lead contamination, the bacteria would cause the water pH to drop and detected with a pH test strip. This project was conceived as a cost effective solution to the Flint water crisis, allowing residents to test if their water was contaminated. This project was created by Eric Borges, Zahra Khazal, Shelby McQueston, Allison Van Fechtmann.
This concept was modified by removing the proton pump idea and instead making the test colorimetric in nature. The plasmid that would be inserted into a bacterial host would now include chromoproteins that would change lead contaminated water different colors to indicate levels of contamination. The plasmid would contain Ppbr promoter sequences that would bind selectively to varying lead particle concentrations and then produce specific colors that would correspond with the contamination level. This concept was chosen in favor of the pH based method due to the difficulty of plasmid construction and lack of published proof of concept.
A second component of the experiment was developed involving a probiotic that would sequester lead and act as an emergency prophylactic. The organism chosen for this part of the experiment was L. rhamnosus, a common probiotic that contains natural lead binding capabilities.
The vectors that were decided to use in this experiment to test the proof of concept of the chromoproteins were the Pet21a and Pet42a vectors. Colors were decided on their ability to mix to form a progressing color scale, moving from a light color to indicate lowest levels of contamination to a dark color to indicate highest levels of contamination. These plasmids would be inserted into E. coli and test colors of interest.
The backbone for the main construct in the experiment was derived from Development and Application of a Synthetically-Derived Lead Biosensor Construct for Use in Gram-Negative Bacteria, in the journal Sensors. The backbone, known as the Pbr-pbr-Rgfp backbone, contains the selected lead promoter pBRr that is necessary to detect lead in the water samples of interest.
This image is from Development and Application of a Synthetically-Derived Lead Biosensor Construct for Use in Gram-Negative Bacteria. It shows that the promoter expressed GFP when lead was present.
The lead promoter was to be mutated using error prone PCR to create promoters with different lead binding affinities. These promoter were to then have chromoproteins inserted downstream that corresponded to the level of lead affinity. RFP served as the reporter for preliminary testing of the pBRr promoter, to test for successfully insertion of the plasmid into E. coli and to see the inducible effects of the promoter. Gibson method was employed for the insertion of the backbone. Several trials were conducted with no successfully transformed clones.
The probiotic being tested, L. rhamnosus, has natural lead binding capabilities and we determined that the technique of force evolving would be the best route to create a emergency prophylactic. This process involves exposing the probiotic to increasing levels of lead to create strains with high lead tolerance. These strains will then be subjected to more rounds of lead testing until a strain that sequester high amounts of lead is achieved. To accomplish this goal we first needed to establish the growth curves of L. rhamnosus to determine the optimal point in development in which to introduce the lead. This was achieved through the incubation of overnight liquid cultures and the testing of optical density to observe growth. For this process, cultures were diluted to an OD of .1 at the beginning of reading. After testing the of growth cycles, the probiotic was then treated with lead concentrations in the liquid cultures. Data from this portion of the experiment showed little change or erratic results. A professor specializing in microbiology on WPI’s campus was consulted, Scarlet Shell, and adjustments were made to made to the process of collecting of optical densities. It was theorized that the dilution to an optical density of .1 was not low enough due to the bacteria growing too fast with the lead having a negligible effect. The optical density was then reduced to .01 to account for this growth. After data still showed irregularities the process was again modified, moving away from liquid cultures to direct plating on agar plates containing varying lead concentrations.
To measure the lead absorbance in water of the L. rhamnosus a colorimetric lead assay was developed. The assay was developed because typical lead-water testing uses equipment that is not present on WPI’s campus and sending out samples for testing is both expensive and not recommended because bacteria is being intentionally seeded in the samples. Multiple companies were contacted regarding colorimetric testing in industry and were consulted when developing the assay. The concept for the assay was derived from a gold nanoparticle heavy metal detection assay from A Fast Colourimetric Assay for Lead Detection Using Label-Free Gold Nanoparticles (AuNPs), originally published in the journal Micro machines. The test was then re-written and subjected to multiple tests to optimize it for low level lead detection. It could not be optimized for our project because the results were too variable to identify lead concentrations in LB and MRS. Instead, a Lead Test Kit from Hach was employed for further testing.