Part:BBa_M50082:Experience

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Applications of BBa_M50082

We ordered this DNA via DNA2.0 and transformed it into E. coli in efforts to induce a biological pathway for salicylic acid production.

Stanford Location

plasmid name: pHDE DNA 2.0 # = 309056 Gabe Barcode: 0133027151 Suzy Barcode: 0133027166 Matt Barcode: 0133021734

Organism: E. Coli Device type: Actuator Box Label: Bioe44 fall 2017

User Reviews

UNIQ49047a40f4725e15-partinfo-00000000-QINU UNIQ49047a40f4725e15-partinfo-00000001-QINU

pH Testing: Our first method for testing the for the production of salicylic acid was through two different types of pH testing. To do this, we took our five different samples at the varying levels of IPTG and let them incubate overnight. The following day, we spun down our samples and tested the pH of the supernatant on pH strips that tested in the 6-8 range. Using a mix of media and IPTG as well as uninduced cells as controls, we ran this experiment 5 times over the course of the 6 week experimental period, with the most effective results being shown in figure 1.

After we using the pH strips, we used a pH probe for a more specific and quantitative testing method. We calibrated the probe and submerged it in the supernatant of each sample until a reading appeared, washing with water between each sample. The results of this experiment, which we ran 4 times over the course of the testing period, can be found on the graph in figure 2.

According to these findings, the SAGD does produce a measurable amount of acid under IPTG induction. The average pH is statistically lower under higher IPTG induction, indicating that the protein production did yield an acidic product. This is confirmed via our negative control in the 0M IPTG induction sample, which compares a standard cell culture to one hypothetically yielding salicylic acid.

Western Blot: After receiving the results from our pH testing, we wanted to verify that a perceived gradient could possibly be attributed to our enzyme. In order to test for a correlation we verified that our desired proteins were indeed being produced by running a Western Blot, using a procedure adapted from Drew Endy’s lab through the fundamentals of bioengineering lab book[4]. According to our research, the pchB protein, which we paired with a HIS tag, had a size of around 13 kilo daltons, while the pchA protein, which we paired with a FLAG tag, had a size of around 55 kilo daltons.[4] The results of our western blot procedure can be seen in figure 3. From this experiment, we can conclude that the proteins are in fact being produced in the E. coli cultures. The western blot shows our target proteins being produced in the expected range according to the standard kD ladder. We also show that under 0M IPTG induction, there is a lack of protein production. This finding is not completely clear due to shifting of samples across lanes in the western blot, however there is certainly a lower amount of protein expressed in the 0M IPTG lanes of figure 3.

Ferric Chloride Testing: Finally, we ran a Ferric Chloride test. Salicylic acid is functionally a phenol ring with an attached carboxylate group. When added to a solution containing phenols, Ferric Chloride reacts to form purple/red complex in solution, providing a general test for the possible presence of salicylic acid. We decided to run the test to determine if there was any reaction at all between FeCl3 and our samples, run against a negative control of media and FeCl3 and five positive controls containing various levels of salicylic acid. Before we ran the test itself, we ran an optical density test of the cells at the 600 nm wavelength. We did this both to have information regarding cell density in our samples so that we could adjust for it following our FeCl3 test results and also, recognizing chorismate as a part of E. Coli’s metabolic pathway being taken away by our pathway, to determine if there may be a correlation between levels of IPTG induction (and this presumably levels of our plasmid working) and cell replication and growth. We found an inverse relationship between our levels of IPTG induction and the optical density. Following an OD600 of our cells, we proceeded to run a Ferric Chloride test on our samples. To do this we put 200 microliters of each sample into three wells for each sample. As a positive control we used solutions with concentrations of .001 M, .00001 M, .0000001 M, 1x10-9 M, and 1x10-11 M salicylic acid. As a negative control we used 200 microliters of media. We added 40 microliters of 1% FeCl3 to each sample, stirring gently with a pipette tip to mix. After mixing, we tested the absorbance to see if there was a detectable change in the different samples being mixed with FeCl3. The results of this experiment can be seen in figure 4 and can be compared to the figure 5 which shows a standard absorbance curve we created using samples of varying levels of salicylic acid and FeCl3.

According to the results shown in figure 4, there is not an obvious result available from the ferric chloride assay. Using a standard salicylic acid concentration as a positive control, we demonstrated that the degree of detection rapidly decreases below a threshold of 1x10-11 M. Therefore, although the assay did not report a positive result indicating the presence of phenols, we can also not rule out its presence.

Conclusion:

It is not explicitly evident from the results collected that the creation of a salicylic acid generating device was successful. However, utilizing pH change as a proxy indicator for salicylic acid production, we have strong reason to believe that the device was at least partially operational. There was a statistically significant drop in pH when comparing the least induced samples at 0 M IPTG to the max induced 0.1M IPTG samples (P value = 0.0259), consistent with our proposed findings. There is also an incremental drop in pH as we increase the IPTG concentration, suggesting that the level of IPTG, and thus protein expression, is related to the pH change. It is important to note however that outside variables do have the potential to affect pH within a cell culture, including processes inherent to cell replication and metabolism. It is therefore necessary for other assays to be utilized for a more conclusive result. To supplement the pH results, we attempted to detect the phenol structure of salicylic acid via a ferric chloride assay, but were unable to confirm the presence of phenols in our samples. While this could be interpreted as an invalidation of salicylic acid production, it is highly probable that our cells were not producing a great enough concentration of salicylic acid to be detected via this assay. To further investigate, we attempted to determine the range of detection using standard salicylic acid concentrations where we confirmed that the assay was unable to detect concentrations of salicylic acid below a threshold of 1x10-11 M. According to the cell concentration in our culture, we would expect the amount of salicylic acid to be around 1x10-15 M, well below the accurate response range.[9] In addition, the ferric chloride assay produced an off target precipitate during testing that only further confounds data collection. OD600 analysis would inadvertently record the unintended precipitate as the intended color change, thus rendering the assay kaput. In a final attempt to collect information about the devices operation, we completed a western blot for two crucial proteins composing the salicylic acid generating device. The results were undeniably positive as it was clearly shown that both proteins were being produced in strong concentrations. While this can not prove that the device was operational, it does prove that the proteins were present and barring any mutations, were likely producing salicylic acid. Since we did not have the capability to extract or purify the salicylic acid directly, it is difficult to definitively state that the device was operational. In future experiments, we hope to purify salicylic acid out of solution so that we can directly quantify the amount of salicylic acid our cells are able to produce. It is also possible that additional laboratory infrastructure such as HPLC, GCMS, or other data collection methods may be able to definitively prove the existence of salicylic acid in follow up tests. The potential ramifications of this project go beyond producing a singular medical product. Along with producing salicylic acid, future directions for the project may include the synthesis of additional molecules via genetic actuators. Products such as Zinc Oxide or retinol A would be beneficial for an all in one facial care product assuming the proper proteins could be utilized. Providing salicylic acid as a proof of concept, it may be one day possible to design a complete medicated facial biome in a convenient, safe, and cost effective manner.