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.
- 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.
The image above, Figure 1, is the last cloning attempt of the PCR chromoprotein inserts, specifically AmilCp and aeBlue. There were no visible colonies on either plate and all other plates were similar in results.
Figure 2 (left) & Figure 3 (right)
Above in Figures 2 and 3 are the Gel electrophoresis runs of digested transformations of pBB-pbr-Rgfp in vectors Psb1a3 (left) and Psb1c3 (right). Experimental vectors were the same size and length on gel as control, uncut plasmids.
The final growth curve of B.subtilis in standard LB medium with a culture starting OD of 0.1 is shown below in Figure 3. The results of each time point are an average of three trials with each trial having three separate cultures all growing under the same conditions. The data show that B.subtilis typically begins to enter logarithmic phase at around 120 minutes and growth begins to slow at around 420 minutes, however growth does continue slightly overnight.
The final growth curve of L. rhamnosus in standard MRS medium with a culture starting OD of 0.1 is shown below in Figure 4. The results of each time point are an average of three trials. Each trial was run with three separate cultures all growing under the same conditions. The data show that L.rhamnosus typically begins to enter logarithmic phase at around 180 minutes and growth continues substantially overnight.
This was our final trial of performing growth curves in liquid culture with varying lead concentrations added to the media. Previous attempts can be found within our notebook, which include starting at an OD of 0.1, lower and intermediate lead concentrations, as well as adding lead at different parts of the growth curve. Focusing on our final trial, we started at a starter bacterial OD 0.01 as advised by WPI microbiologist Dr. Scarlet Shell along with picking specific lead concentrations that literature predicted would show a noticeable effect on the growth of both B.subtilis and L.rhamnosus. The specific lead concentrations were added after being diluted back to 0.01 and before lag phase began. As seen below in Figure 5, B.subtilis growth slightly inhibited at 400 ppm beginning at 150 minute time point and can see a progress of lag behind both the control and 100 ppm which grew hand in hand. A more major decrease in growth by measure of OD can be noted by the culture growing in 800 ppm of lead.
The same inhibition was not seen with our lead concentrations picked in L.rhamnosus. L.rhamnosus starting culture was diluted back to 0.01 OD before introducing the distinct lead concentrations within their lag phase. As seen in Figure 6, no real conclusions can be drawn due to their is no inhibition of growth in up to 50 ppm of lead. This is one of our several reasons we decided to not continue future experiments with L.rhamnosus because of non-promising results and also with the pressure of time, but to focus on B.subtilis which most importantly has the ability to bind more lead than L.rhamnosus.
Lead Contaminated Agar Plates
Figures 7 and 8 , shown below, show B.subtilis growing on a control plate compared to B.subtilis growing on plates composed of 1000 ppm, 1500 ppm, and 2000 ppm lead. The plates in Figures 7 and 8 were made by streaking one colony from an LB control plate onto the plates shown below. The number of individual colonies counted on each plate is written on the plate. Our hypothesis was that the higher exposure to lead, a decrease in colony count number could be noted. Results from this experiment showed our data was not consist with our hypothesis. However, colony diameter on the 1500 ppm and 2000 ppm lead plates seen in Figure 8 decrease as the colonies are exposed to more lead. This data aligns with our hypothesis.
Figure 7 (left) & Figure 8 (right)
Curious to whether or not these bacteria were just tolerating the lead on the plate or actually binding it, we decided to look under a microscope and compare morphologies between the varying concentrations. B.subtilis is seen in the series of photos below(Figures 9, 10, and 11), as lead concentrations increase from 0 ppm (control) through 2,000 ppm, B.subtilis colonies begin to form a ring around each colony. The colonies also begin to appear more fuzzy as lead concentrations increase. We hypothesize that this ring is due to dead cells within the colony and the fuzzy appearance is the result of lead that has bound to the surface protein of each individual B.subtilis cell within the colony. However, the colonies in the 2,000 ppm seen in Figure 3 look different from every other morphology. We hypothesize that this could be because they are no longer B.subtilis or because the bacteria have evolved to live in the high lead concentration. L.rhamnosus microscopic analysis were not promising, but can be seen within our notebook.
Figure 9 (left), Figure 10 (middle), Figure 11 (right)
Figure 12, shown below, shows data from the experiment described above that was observed with L.rhamnosus. On the left is a control plate streaked with L. rhamnosus and on the left is a 500 ppm lead plate streaked with L.rhamnosus. The response to L.rhamnosus to high concentrations of lead is similar to B.subtilis seen in Figures 7 and 8 in that the colonies become smaller and decrease in number.
While meeting with WPI’s microbiology Professor, Scarlett Shell, our team adapted our protocol in an effort to gather more quantitative data regarding bacterial response to lead contaminated plates. The new protocol involved rolling liquids cultures with known ODs onto lead contaminated plates. Theoretically, a known OD used to roll onto plates would give relatively consistent colony numbers on each experimental control plate. Numbers of colonies on lead contaminated plates could then be compared in a ratio-like fashion to the control plate for that experiment.
The first step in this protocol is finding the OD which would be used to spread onto plates which would create a consistent number of countable colonies. Table 1, shown below, shows the measured ODs of each liquid culture for B.subtilis and L.rhamnosus before it was plated onto the control plates as seen in Figure H. The spectrometer used to read OD of these cultures is unable to read ODs lower than 0.01, as a result the 1:10,000 dilution has an estimated OD of 0.001.
Below, Figure 13 shows the results of an experiment where multiple serial dilutions with measured ODs were plated in order to count the colonies. In addition to the dilutions indicated above in Table A, both 1:1,000 dilutions and 1:10,000 dilutions were plated using 10uL of culture and also using 90uL of culture. These results of these plates can be seen in Figure 13.
As seen above, the number of colonies growing on every plate are overgrown. This shows that the starting ODs measured and shown in Table 1 are too dense to plate and obtain countable colonies. Due to the fact that the spectrometer was unable to read ODs smaller than 0.01, we decided to estimate OD at greater serial dilutions by diluting further with a solution with a 0.01 OD.
Figure 14, shown below, shows a schematic of an experiment intended to find a dilution of overnight cultures of B.subtilis and L.rhamnosus that could give consistent and countable colonies on control plates before plating that dilution onto varying concentrations of lead. The experiment depicted in Figure 14 was designed to determine whether a 1:10,000 dilution or a 1:1,000,000 dilution would provide a countable number of colonies on each plate. The experiment was also designed to determine the amount of variability between two plates plated cultures of the same OD.
The experiment shown on Figure 14 was performed with both B.subtilis and L.rhamnosus. Each culture OD was measured after it was diluted 1:1,000 to ensure that the OD was 0.01, and it was further diluted from that point. Figures 15 and 16, shown below, indicate the number of colonies that grew on each plate.
Figure 15 shows that plates A1, A2, B1, B2, C1 and C2 all grew a number of colonies which could be counted definitively. All of these plates were plated with a 1:1,000,000 dilution of B.subtilis. Despite the fact that each of these plates should ideally the same number of colonies, there is variation. This variation exists mostly between plates B1 and B2 as well as C1 and C2.
Figure 16 shows that the 1:1,000,000 dilution of L.rhamnosus grew more colonies than the same dilution of B.subtilis. It also shows that the data for L.rhamnosus was far more variable than the data for B.subtilis. This, along with that data from L.rhamnosus growth curves in lead which showed no response, allowed us to decide to continue our probiotic testing with only B.subtilis. In order to observe how varying concentrations of lead effected grown of 1:1,000,00 serial dilutions of B.subtilis, they were placed on four different concentrations of lead plates.
Figure 17, shown below, shows 1:1,000,000 dilutions of B.subtilis growing on control, 50 ppm, 200 ppm, 1000 ppm, and 1500 ppm.
Table 2, shown below, shows the number of colonies counted on each of the plates along with the calculated average between the two plates for each lead concentration.Table 2, also shows the ratio of the average number of colonies for each lead concentration in relation to the control in the form of a percentage.
The data in Table 2 shows that that the number of colonies growing on each concentration does not align with our hypothesis that increased lead concentration would decrease the grown of B.subtilis. We decided to move forward by not focusing on the inhibition of growth to show lead binding but to use the Hach Lead Assay to quantify B.subtilis colonies lead binding abilities from both 800ppm and 2,000ppm plates.
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.