Difference between revisions of "Team:Santa Clara/Results"
Rkousnetsov (Talk | contribs) |
Rkousnetsov (Talk | contribs) |
||
| Line 6: | Line 6: | ||
<h1>Results</h1> | <h1>Results</h1> | ||
| − | <p> | + | <p>How did we test?</p> |
| + | <p>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. 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.</p> | ||
| + | <p>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.</p> | ||
| + | |||
| + | <img alt="Initial trial" src="" /> | ||
| + | |||
| + | <hr/> | ||
| + | |||
| + | <p>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.</p> | ||
| + | |||
| + | <img alt="Second trial" src="#" /> | ||
| + | |||
| + | <hr/> | ||
| + | |||
| + | <p>On 9/16/15 we finally were able to clone CFA downstream of the pLac promoter using the Gibson Assembly.</p> | ||
| + | |||
| + | <p>Colony PCR was then used to screen for recombinants.</p> | ||
| + | |||
| + | <img alt="Gel colonies" src="#"/> | ||
| + | |||
| + | <p>These colonies were then used to generate overnights as seed cultures for the acid shock test. The results of this test are shown below.</p> | ||
| + | |||
<img alt="OD Graph" src="https://static.igem.org/mediawiki/2015/9/9b/SCU_iGEM_AcidShockGraph.png" style="width: 100%;"/> | <img alt="OD Graph" src="https://static.igem.org/mediawiki/2015/9/9b/SCU_iGEM_AcidShockGraph.png" style="width: 100%;"/> | ||
| − | |||
<ul class="pager"> | <ul class="pager"> | ||
Revision as of 03:46, 19 September 2015
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. 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.
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.
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.
