Team:Santa Clara/FutureProspects
Future Prospects
Alterations To The Acid Shock Assay
Being as the cloning was successful only very recently, we have not had the opportunity to run all of the assays that we had intended to. Our results indicate that there is in fact a change in the OD readings when the cells are allowed ample time to reenter log phase. The major problem with the previous test is that it was still far too acidic. Even though we saw that there was complete cell growth at a pH of 4, we need to run another preliminary assay increasing the resolution between pH of 3 and 4 to find the critical point at which the cells begin to die off. To do this we will run the same protocol as before but with more samples at different pH between 3 and 4 to better understand the organisms tolerance. Once that critical point is determined we would run the same assay, keeping the pH set slightly below that critical point. This would hopefully yield more conclusive results.
Another alteration we would like to test is to try and pH the solution differently than we did in the media used in the aforementioned assays. We had used HCl as a way pH the solution but we have no way of knowing whether or not it is the acidity that is killing the cells or the chloride ions. Being as we want to replicate the environment of a bioreactor we figure that it would be more applicable if we used the metabolic acids that the cell would be exposed to, to pH our culture rather than HCl. This could be done either by allowing a culture to reach stationary phase then spinning down the cells and siphoning off the supernatant as the broth for the assay or just adding in organically synthesized weak acids, such as lactic acid, butyric acid, etc.
Stationary Phase Assessment
The other test that we would like to run is a simple time course at a pH of 7 comparing both induced and uninduced to see if it might be possible to see a higher OD reading for the induced culture when it enters into stationary phase. Our backing for this hypothesis is that one of the major signaling molecules that push organisms into stationary phase is the extracellular acid that is built up from the surrounding cells. So if the cells become less sensitive to acid, i.e. less can pass through the membrane, then it should require a high concentration of extracellular acid to cause the cell to enter into stationary phase. This, if true, could be incredibly beneficial for bioreactor technology as it would allow cells to reach a higher cell density which would mean larger overall yields
Going from theory to practice
If these test were to show a clear effect from the addition of CFA, our next step would be to move to a larger culture size. We would run a batch culture in a chemostat and monitor the pH as the culture progressed while also monitoring OD readings. We would run this side by side with the induced and uninduced cultures in order to see if the cultures continued to show a higher cell density before entering into stationary phase. We could then also shift to a fed batch system to further simulate the continuous culture system by removing portions of the culture and feeding it fresh media to see if when left in culture over extended periods of time, we can still see the positive result from the expression of CFA.