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From 2014.igem.org

BYU 2014 Notebook Edit September October

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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 Concentration
1 #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 Old Full Strength, 10e-1, 10e-2, 10e-3.
2 #1 new Full Strength, 10e-1, 10e-2, 10e-3.
3 #7 old Full Strength, 10e-1, 10e-2, 10e-3.
4 #7 new Full Strength, 10e-1, 10e-2, 10e-3.