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BYU 2014 Notebook Edit September October

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Michael linzey 9/8/2014
-I took a sample of the CRISPR mutagenisis and streaked it out on another plate
-This will allow us to have spare plasmid to test our mutated CRISPR system
-I left the plates in overnight to allow them to grow up
-Helped Garrett prepare for the intial CRISPR tests

Michael Linzey 9/9/14
-I used the plasmid purification kit to extract the mutated plasmid
-I prepared a sequencing test so we could see if a mutation had taken place

- 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 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.





 Michael Linzey 

-We worked as a team and set up the CRISPR test

-So I was involved in everything that Garrett talked about above 

 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!



 Michael Linzey 9/15/2014 

-I got the sequencing results back today and I spend an hour or so trying to figure them out

-I consulted with Dr. Grose and we determined that the forward primer didn't work and I needed to redo the experiment

-In order to do this I was going to need more CRISPR plasmids that had their restriction sites mutated. Luckily I had streaked the bacteria onto new plates so I had ample bacteria to use. I then used the Plasmid Purification Kit to extract the purified plasmid. 
   

 Garrett Jensen - 9-16-2014

 - Yesterday the CRISPR would not grow up. I inoculated the LB/Amp/Chloro broth twice but it never grew up. I thought it might be the broth so I tried growing it up in just LB/Chloro broth. Today that had worked. It seems a little weird that the CRISPR would grow up on a Chloro/Amp plate but not in the broth. I think that something is wrong with our broth. So today I made up a new batch and I am trying to grow up our CRISPR again. 

 - Today I did plate the CRISPR that grew up and tested it. I have spot tested 5 µL of T7 dilutions from 10e-3 -> 10e-10. As a control I have run Dh5a and the empty iGEM plasmid pSB1C3 with T7 phage infection to ensure that the plasmid is not conferring phage resistance. Cross your fingers!!!



Michael Linzey 9/17/2014 

-Today I spent some time preparing plasmids for sequencing. I put two ul of plasmid with one ul of a forward or reverse primer. I brought these up to another lab where they will be put through a DNA sequencer. This will allow us to see if the mutations worked. 

- I also helped Garrett set up the next round of test for the CRISPR system, this mostly included trying to understand the results that we got and setting up another spot test.