Results

Biosensor Results

Initial trials of gibson assembly were conducted for chromoprotein inserts into Pet21a and pet42a vectors. Several iterations of both gibson assembly, PCR, and transformation into chemo-competent E. coli were conducted. Our first Gibson assembly utilized the six reconstituted chromoprotein genes (no PCR), miniprepped and purified pET21a and pET42a vectors, and chemo-competent DH5-a E. coli. Since we didn’t run a PCR for the chromoproteins, we checked to make sure the DNA concentrations were acceptable for the assembly (table? 6/27); the results were positive at ~10 ng/ul for each gene. Blue chromoproteins were assembled into pET21a, yellow and red genes were assembled into pET42a. Eight transformations were performed, with two negative controls - non-transformed E. coli streaked onto ampicillin and kanamycin plates. Only the three plates with pET21a grew colonies, including the negative control, while no colonies grew on any pET42a plates. This indicated that the transformation was unsuccessful, but we were confused why colonies grew on the negative control for ampicillin. To understand why, we streaked more susceptible E. coli onto ampicillin and kanamycin plates. After 24 hours, only ampicillin plates grew colonies, indicating that none of our ampicillin plates had the proper concentration of antibiotic to inhibit growth of susceptible bacteria. We re-made the LB-ampicillin plates for subsequent transformations. We again tried the Gibson assembly. The assembled products were run on a polyacrylamide gel and appeared to be successfully assembled and of the appropriate size. Gel purification and transformation of constructs into E.coli was attempted but efficiency was low, indicating unsuccessful transformation. Transformation with PCR products was attempted a second time but transformation efficiency was still low. PCR of chromoproteins was attempted again. The purified PCR products were used for assembly and transformation into E. coli yet again and plates were left in the incubator at 37C for overnight and did not yield expected results.

Experimental differences

• Reconstituted chromoproteins tried first (not PCR-amplified)
• PCR-amplified chromoproteins and used products for gibson without PCR cleanup
• Cleaned-up PCR products and tried again several times
• Extended Gibson product thermocycler incubation time from 30 mins to 45 and 60 mins
• Tried different vectors: pET21a & pET42a, psb1c3 & psb1a3
• Used SOC and LB for cell recovery after Gibson
• Restriction digest cloning was attempted
• Extended B. subtillis & E. coli incubation in SOC (?) and on plates (48 hr)
• Checked, revised and modified chromoprotein PCR primers — initially had universal primers for all 6 chromos but made individual primers specific to each gene. Modified the individual primers to anneal more sequence

After the unsuccessful attempt during the summer to clone the chromoprotein inserts into B. subtilis and DH5-a E.coli, efforts were redirected to cloning the pBB-pbr-Rgfp construct into the vectors Psb1c3 and Psb1a3. At first the pBRr promoter was attempted to be cloned and PCR’d but this yielded inconclusive results. Minipreps of the vectors Psb1c3 and Psb1a3 were conducted and the construct was inserted into the vectors via Gibson Assembly method. The ligated construct was then transformed in competent E.coli and plated on chloramphenicol (CAM) LB agar plates. When compared against control plates, plates being tested for the insert had a very low efficiency or did not have visible colonies for either vector. A second miniprep was conducted of the vectors and the assembly was repeated. When transformed, a few plates yielded more colonies than the previous round. These colonies were minipreped, digested, and run on a gel to confirm successful transformation. When compared to control vectors without inserts added, the transformed vectors were of the same size indicating unsuccessful transformation. This indicated that the E.coli most likely took the vector but either kicked out the insert or the insert was never properly assembled into the plasmid. A final third attempt following the same procedure was conducted and the same results were achieved, with a low transformation efficiency for both vectors.

Probiotic Results

Lead Assay Results

The goal of the lead assay was to have a colorimetric assay that we could use to measure how much lead L. rhamnosus and B. subtilis was actually removing from water. It was needed because other lead-water testing uses equipment we do not have access to and sending samples for testing is both expensive and not recommended because we are intentionally putting bacteria in our samples. The assay was read in a plate reader in a 96-well plate. The absorbance was the result of interactions between glutathione, 20nm gold nanoparticles, and lead. The solution started a light pink color, and as an increasing amount of lead was present the solution would change to a darker purple-blue color. Before deciding that this assay would not be appropriate for our project, we did extensive assay development experiments to try to address its variability. We tried different protocols, optimized wavelength on available machinery for water, MRS, and LB, optimized GSH concentration for water, MRS, and LB, optimized pH of the solution and of the phosphate buffer for water, MRS, and LB, tried various lead concentrations, optimized type of gold nanoparticles, optimized how dilutions were made, optimized making of the GSH solution, and optimized reading time frames. In addition to this, we also found that readings were more accurate when the assay was done row by row, the gold nanoparticles were kept cold, and when the GSH and gold nanoparticles were added within 20 seconds of one another. We tried vortexing the samples before reading them; we double and triple checked the math for each dilution, and we considered doing a standard curve each time as if it were a Bradford Assay. Despite all of our work, the standard curve that was developed was not stable enough to accurately determine the concentration of known-unknown samples, and the assay could not be used in our project.

One of our standard curves can be seen below:

This graph shows one of our preliminary standard curves. Absorbance is on the y-axis, and lead concentration in parts per billion is on the x-axis. It shows that as the lead concentration increases so does the absorbance. Four samples were read per point on the graph, and the average of the four was used in the graph. The error bars on this graph looked very promising because of the limited overlap.

This is the same graph as the one above. This shows one of the reasons this assay was proved to be ineffective for our project. Instead of averaging all four of our samples per concentration, we averaged the three points that were closest for each lead concentration. The error bars increase because the difference in absorbance is not significant enough between the different lead concentrations. Similar results were seen throughout our assay development. This graph shows why we were unable to place other samples of known lead concentrations on the standard curve without looking at the concentrations.

Lead Test Kit Results

After our failed attempt at creating a DIY lead assay, we chose to use the Hach LeadTrak kit to quantify the amount of lead that L. rhamnosus and B. subtilis could remove from water. The protocol involves running a sample through a Fast Column Extractor, adding a lead indicator to the eluent, and measuring the OD477 of the sample. After creating a standard curve using the lead standard solution provided in the kit that plots lead concentration (up to 150 ppb) against absorbance at OD447, the lead concentration of any sample can be determined. Our standard curve is shown below:

Using our standard curve, we were able to quantify the amount of lead absorbed by our “force-evolved” B. subtilis culture over a 24-hour period. As previously mentioned, B. subtilis was streaked on a LB-agar plate that contained 2,000 ppm of lead. We hypothesized that because the B. subtilis colonies on this plate had grown in the presence of an extremely high quantity of lead, they would would have evolved to bind more lead than colonies that had grown without any lead (control colonies). In order to determine whether the “force-evolved” culture could in fact bind more lead that the control culture, we measured the lead absorbance over time for cultures made from each of the two colonies. The colonies used to produce the control culture were taken from a standard LB-agar plate streaked with B. subtilis. The second, “force-evolved” culture was produced using colonies from the 2,000 ppm lead LB-agar plate. Each culture was grown and sampled in a accordance to the Lead Test Kit Protocols provided on the Experiments page.

The amount of lead bound to B. subtilis for each culture over time is shown below:

In order to ensure that the difference in lead binding over time was not a result of differences in the number of cells present in each culture (determined by the OD600 of the culture), we control for this by dividing the lead concentration by the OD600 in the graph below:

We believe that the large amount of error associated with the first time point is due to the fact that the OD600 of the cultures at this point is still very low. Once the cells reach log phase (hours 4 and 24), we can see an increase in lead binding per OD overtime with minimal error, suggesting that the “force-evolved” culture could have bound more lead over time than the control culture.