Team:Santa Clara/Results
Results
How did we test?
In order to isolate the action of the CFA synthase within our system we wanted to see the acid resistance of a cell while none of its other defense systems were in play. To do this we studied the typical acid regulators and how their transcription is initiated. In E. coli specifically the vast majority of its acid resistant genes, including CFA, is under the control of the RpoS sigma factor which is induced under acid stress, which typically occurs in the stationary phase of cell growth. With this in mind we decided to run a acid shock assay to put the put the cells in an acidic condition without being able to buildup its resistance ahead of time.
While attempting to build our plasmids, we wanted to try and standardize the acid shock procedure so that once we successfully created recombinants we could run the assay after the problems had been solved. Below is the data from our acid shock test.
Initial Acid Shock Assay of T7 Expression E. Coli. Negative time points refer to the time in which acid shock was initiated, 0 is immediately after acid shock, and then time points were taken every 30 minutes. Cells were continually cultured at a pH 3 at 37˚C with shaking at 250 rpm.
We saw that we were getting complete cell death after one hour of exposure to the pH 3 media but we wanted to be sure that it was not too acidic. If it was then even with the aid of our resistance system we would not be able to see any change in tolerance. The graph below is a replication of the prior assay but using a spectrum of pHs.
Range of pH survival of untransformed T7 expression E. Coli. Negative time points refer to the time in which acid shock was initiated, 0 is immediately after acid shock, and then time points were taken every 30 minutes. For pH 7, time 210 minutes the OD reads over 1, this is falls out of the pattern for that condition and has been ignored in data analysis. The average between two replicates is represented. Cells were continually cultured at a pH 3 at 37˚C with shaking at 250 rpm.
On 9/16/15 we finally were able to clone CFA downstream of the pLac promoter using the Gibson Assembly.
Colony PCR was then used to screen for recombinants.
These colonies were then used to generate overnights as seed cultures for the acid shock test. The results of this test are shown below.
Acid Shock Assay of pLac +CFA mutants. Acid shock was done with LB at a pH of 3. Cultures were induced at 60, 30, 15 minutes before, and at acid shock with 1mM IPTG. IPTG concentrations were maintained through acid shock. Cells were continually cultured at a pH 3 at 37˚C with shaking at 250 rpm.
The above graph shows that after acid shock there is a difference between the samples induced for 0-15 minutes, 30-60 minutes, and not at all. The increase in OD falls in suit with what was expected because it was anticipated that the CFA induced cultures would act in a similar trend as seen when the assay was run across several pHs. In Figure 2 there is an inverse correlation in the OD readings at stationary phase and the levels of acidity, as in the more acid the lower the OD. Based on this trend it would appear as though the induced cells are able to tolerate the acidity as if it was uninduced at a higher pH. This would elude to CFA having the phenotypic effect of increased acid resistance that we were looking for. However we wanted more conclusive evidence as OD readings merely measure the amount of debris blocking the path of light and indicate nothing about cell vitality. So to test cell vitality we plated the culture at each time point to see growth as a test for cell viability. The plates came back with lawns at the initial time point prior to acid shock and then no growth there after for all cultures. This leads us to believe that for that aspect of the procedure we will need to tune the pH of the acid shock to ensure that the cells can survive if helped by CFA. This will be discussed more extensively in the following section.