Team:WLC-Milwaukee/Demonstrate
Results
Cloning
Our cloning results were confounding initially, however, we were able to obtain what appeared to be successful clones of our parts in pTrc99a as indicated by diagnostic restriction digests. Clones screened in this manner were sent for sequencing and we received some positive sequencing results.
This data led us to believe we had our parts successfully in pTrc99a allowing us to continue with our cloning as laid out on our Wet Lab page.
We continued our cloning using primers designed to amplify both our parts and the trc-promoter from the pTrc99a backbone as the part BBa_K2042004 did not have samples in the registry. We were able to successfully amplify our parts, and select potentially successful clones using diagnostic digests. Several of our positive clones were also sent for sequencing.
We also performed Native SDS PAGE Gel electrophoresis stained using Coomassie staining seen below. These results attempting to confirm protein presence were largely inconclusive and nearly all bands on the PAGE gels appeared light and washed out.
The X-short J-protein from Lambda phage with a C-terminal His-tag was expressed with the pTrc99A promoter and a purification attempt was made using a Qiagen Ni-NTA spin kit. Samples were run on a 10% PAGE gel and stained with Coomassie blue stain. The red box is around a band that may correspond to the target purified protein. The J-protein fragment should be approximately 28 kD.
The J-protein from Lambda phage with an N-terminal His-tag was expressed with the pTrc99A promoter and a purification attempt was made using a Qiagen Ni-NTA spin kit. Samples were run on a 10% PAGE gel and stained with Coomassie blue stain. The red box is around a band that may correspond to the target purified protein. The J-protein fragment should be approximately 28 kD.
We then proceeded to various Horseradish peroxidase experiments to better design and optimize our kit.
HRP Results
Our testing of Horseradish peroxidase (HRP) began with measuring the absorbance of HRP in hydrogen peroxide (H_2 O_2) at 408 nm using UV vis spectrometry. We added HRP in small aliquots to an excess of H_2 O_2. This test yielded very small absorbances, but nonetheless showed an upward trend, which was expected. We expected the solution to turn red, but that did not happen, so we transitioned to other testing.
The next phase of testing involved chemiluminescence. A 10% hydrogen peroxide solution was made volumetrically and brought up to volume with pH 7.9 1X PBS (phosphate buffered saline). A ~500X luminol solution was made using 0.0145g of luminol and 1 mL of DMSO. 2 mL of the hydrogen peroxide solution with 20 uL of the luminol solution served as a control because it showed how much light was emitted from that without any HRP. Tests were done with 1, 2, and 3 uL of 1 ug/mL HRP added to a control solution. Each sample was run for 30 minutes, and two replicates were done of each sample, to end with 3 trials for each set of data. A consistent max light emission was observed between 5 and 10 minutes, making this the optimal measuring range for use in a test kit.
Here is an example of a fluorometer graph from Trial 1 of 1 uL of HRP
Averages were taken every 150 second interval.
The next stage of testing involved using tetramethylbenzidine (TMB). TMB, used in conjunction with HRP, yields a colored product. When HRP is added, the solution turns blue, then green, and finally yellow. The blue product is measured at 652 nm and the yellow product is measured at 450 nm. 1 X TMB was diluted to a 5% solution with diH2O. The 100 ug/mL HRP solution was diluted to a 2% solution, and varying amounts of that were used. 600 mL of TMB was used for each trial with 2 uL, 5 uL, or 8 uL of HRP. The uv vis of the solution was taken every ~0.5 seconds. The graph below shows how an increase in HRP increases the speed of the reaction, allowing it to reach the maximum absorbance at 652 nm sooner.
Initial Proof of Concept Testing
Water samples were prepared by growing overnight cultures of wild-type (WT) E. coli (MG1655) and an E. coli strain with a lamB gene deletion. The J-protein from Lambda phage interacts with the LamB protein in order to bind to E. coli. Therefore, the loss of the lamB gene should result in the J-protein not binding and serve as a negative control. The WT and lamB deletion E. coli cultures were either not diluted or diluted 1:100 up to four times to generate 10-2, 10-4, 10-6, and 10-8 dilution samples.
Prior to preparation of the water samples, the J-protein with an N-terminal His-tag was purified. The horseradish peroxidase (HRP) enzyme was then attached using an HRP conjugation kit purchased from Abcam (AB102890). Approximately 25 microliters of the J-protein-HRP was added to 10 milliliters of each water sample containing E. coli. Additionally, 25 microliters of J-protein-HRP was added to sterile water to serve as another negative control. An additional negative control consisted of only sterile water with neither E. coli or HRP added.
Each 10 ml water sample would be poured into a syringe attached to the filter apparatus. The water would then be pushed slowly through the 0.22 micron pore size filter (catches the bacteria) with the plunger. An additional 15 ml of sterile water would then be pushed through the filter to wash through any small material or unbound J-protein-HRP. Because the adaptor to hold the filter we ordered did not arrive before we needed to run the tests, we used a vacuum filter unit with the same filter disks we would have used in the syringe and adapter.
The filter paper was then removed from the filter apparatus and placed into a 5% solution of Tetramethylbenzidine (TMB) for two minutes. TMB produces a blue color when exposed to HRP. If exposed long enough to HRP or if large quantities of HRP are present, the TMB will change to a yellow color. Immediately after removing the filter paper from the TMB solution, the color was measured in a UV-Vis spectrophotometer. Blue and yellow colors were measured at 652 and 450 nm respectively. A positive control was prepared by adding 25 microliters of J-protein HRP to a filter without washing and placed in the TMB solution.
The color of the TMB is observed in the small dishes below after the disk was removed. As expected there was no color change when only sterile water was passed through the filter. Also, as expected, the color changed dramatically to a yellow-orange color when the J-protein-HRP was spotted on the filter paper and not washed off.
The remaining data suggests that the J-protein-HRP is not binding specifically to the E. coli cells. The WT and lamB deletion dilution samples look very similar to the sample containing only the J-protein-HRP and sterile water (no cells). This suggests that a small amount of J-protein-HRP is binding to the filter paper itself and that this protein is giving the color change that we are observing in all the samples with bacteria. Interestingly, the most concentrated bacterial samples (WT and lamB) showed the smallest color change. We are uncertain why that is but hypothesize that there are so many bacterial cells on the filter paper, they are blocking the J-protein-HRP from binding to the filter. Therefore the J-protein-HRP is being washed off and a much lower amount of color change is observed.
While it is disappointing that we were unable to get results that showed specific binding of the J-protein-HRP to E. coli cells, there are many ways we could optimize the experiments to potentially improve our results. These experiments were performed by adding an arbitrary volume of J-protein-HRP to the water samples. It is possible that more or less J-protein-HRP would have been better to observe E. coli presence. Also, the stability of J-protein-E. coli interactions have not been examined to determine how well they occur in water. The interaction may require a specific pH or salt concentration. Finally, the timing of the filter paper exposure to TMB has not been determined as well. We chose two minutes because that is when we started to visually observe a color change in some samples. It is possible that additional time may provide more information on how much J-protein-HRP is bound. Future work in these directions may give much more promising results and allow for a viable water testing apparatus.
