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Team:BYU Provo/Notebook/CRISPR/septoct
From 2014.igem.org
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<br/> 31 B test Spot test new. Full Strength, 10e-1, 10e-2, 10e-3. | <br/> 31 B test Spot test new. Full Strength, 10e-1, 10e-2, 10e-3. | ||
<br/> 32 B control control | <br/> 32 B control control | ||
| - | <br/> 1 | + | <br/> 1 #1 spot test Old Full Strength, 10e-1, 10e-2, 10e-3. |
| - | <br/> 2 | + | <br/> 2 #1 spot test New Full Strength, 10e-1, 10e-2, 10e-3. |
| - | <br/> 3 | + | <br/> 3 #7 spot test Old Full Strength, 10e-1, 10e-2, 10e-3. |
| - | <br/> 4 | + | <br/> 4 #7 spot test New Full Strength, 10e-1, 10e-2, 10e-3. |
| + | </pre><br/><br/> | ||
| + | </p> | ||
| + | <p><u> Garrett Jensen - 9/11/2014</u> | ||
| + | <br/> - Our plates had some interesting results. The controls did not grow, but that was because we plated both strain B and DB5a on Cam+Amp plates. The dilute phage plates did not have much growth. T7 phage pretty well wiped out the E. coli. The spot test plates also had big plaques. The interesting result was that there were many colonies growing inside of the plaques on every single plate. <br/> - The lack of CRISPR function could be attributed to a number of different things: We could have designed the T7 spacer region wrong, the T7 phage we used may have a mutation where we chose our spacer, the CRISPR could be non functional. | ||
| + | <br/> - My hypothesis to explain these colonies is that they grew up from the cells that managed to create their own spacer for T7. This type II CRISPR is adaptive and a possible explanation for our results is that it did adapt to the phage infection. | ||
| + | <br/> - The way for us to tell if this is true is to redo the experiment using E. coli from these colonies and run DH5a with the empty iGEM plasmid pSB1C3 as a control. If we do not get plaques on the first and do not see similar results to our last test then we can be confident that our CRISPR is working! | ||
| + | <br/></p> | ||
Revision as of 19:40, 15 September 2014
BYU 2014 Notebook |
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- Garrett Jensen. 9/10/2014
- On Monday we decided to focus on testing the effectiveness of our CRISPR system as well as performing mutagenesis of our CRISPR so that we can submit it to the iGEM registry. These are our priorities as we have to submit the part to iGEM by mid october.
- Today we performed a 1:10 serial dilution of E coli phage T7 out to 10e-8. We will use this on Wednesday to test the effectiveness of our CRISPR system in E. coli. We are not completely sure if T7 will infect E coli Dh5a so we are going to test that as well in comparison with its normal host strain, E. coli B.
- Using the plates we have streaked our E. coli on that contains the CRISPR and the T7 spacer we started overnight bacterial cultures using 20 mL of LB broth with the antibiotics ampicillin and chloramphenicol. I also started overnights of DH5a and B. The CRISPR containing microbes are labelled 1 and 7.
Today I made freezer stocks from our E. coli to preserve the strain that has the CRISPR for future use. They are in Cryo tubes in out iGEM group freezer box.
- Today we plated the E. coli and incubated the T7 phage with the host as well as several tests where we have done a spot test on the E. coli. The Table below details what strains we used, what concentrations of phage were used, and what type of test was performed.Plate Label CRISPR Set T7 Phage Set Concentration1 #1 original 1.00E+00
2 #1 original 1.00E-01
3 #1 original 1.00E-02
4 #1 original 1.00E-03
5 #1 original 1.00E-04
6 #1 original 1.00E-05
7 #1 new 1.00E+00
8 #1 new 1.00E-01
9 #1 new 1.00E-02
10 #1 new 1.00E-03
11 #1 new 1.00E-04
12 #1 new 1.00E-05
13 #1 CONTROL
14 #7 original 1.00E+00
15 #7 original 1.00E-01
16 #7 original 1.00E-02
17 #7 original 1.00E-03
18 #7 original 1.00E-04
19 #7 original 1.00E-05
20 #7 new 1.00E+00
21 #7 new 1.00E-01
22 #7 new 1.00E-02
23 #7 new 1.00E-03
24 #7 new 1.00E-04
25 #7 new 1.00E-05
26 #7 CONTROL
27 DH5a test Control
28 DH5a Test. Spot test new. Full Strength, 10e-1, 10e-2, 10e-3.
29 Dh5 control. Spot test old. Full Strength, 10e-1, 10e-2, 10e-3.
30 B test Spot test old. Full Strength, 10e-1, 10e-2, 10e-3.
31 B test Spot test new. Full Strength, 10e-1, 10e-2, 10e-3.
32 B control control
1 #1 spot test Old Full Strength, 10e-1, 10e-2, 10e-3.
2 #1 spot test New Full Strength, 10e-1, 10e-2, 10e-3.
3 #7 spot test Old Full Strength, 10e-1, 10e-2, 10e-3.
4 #7 spot test New Full Strength, 10e-1, 10e-2, 10e-3.
Garrett Jensen - 9/11/2014
- Our plates had some interesting results. The controls did not grow, but that was because we plated both strain B and DB5a on Cam+Amp plates. The dilute phage plates did not have much growth. T7 phage pretty well wiped out the E. coli. The spot test plates also had big plaques. The interesting result was that there were many colonies growing inside of the plaques on every single plate.
- The lack of CRISPR function could be attributed to a number of different things: We could have designed the T7 spacer region wrong, the T7 phage we used may have a mutation where we chose our spacer, the CRISPR could be non functional.
- My hypothesis to explain these colonies is that they grew up from the cells that managed to create their own spacer for T7. This type II CRISPR is adaptive and a possible explanation for our results is that it did adapt to the phage infection.
- The way for us to tell if this is true is to redo the experiment using E. coli from these colonies and run DH5a with the empty iGEM plasmid pSB1C3 as a control. If we do not get plaques on the first and do not see similar results to our last test then we can be confident that our CRISPR is working!
