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BYU 2014 Notebook Edit July August

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27 June 2014

Alpha Amylase: Added DpnI to the PCR product for the digest of any plasmid that was not mutated properly and placed it in the 37 degree incubator. We will transform this digested plasmid into DH5a tomorrow. (JB)

30 June 2014

Alpha Amylase: Today the colony 8 PCR product for the Alpha Amylase mutant was transformed into DH5a using the basic transformation protocol as listed in the “Protocols” page. Tomorrow colonies with mutant Alpha Amylase pSB1C3 that survived the digest will be isolated and grown, then overnights can be made from these colonies for further work.

I also did some research for grants that we could apply for in order to receive more funding. Below are some links that may be useful to the team and others. (JB)

http://www.werf.org/i/Funding/Open_RFPs/a/o/rfp.aspx?hkey=05bda2a1-23af-4891-badf-815b2960d4f3

http://www2.epa.gov/education/environmental-education-ee-grants

http://www.nifa.usda.gov/fo/waterquality.cfm

https://experiment.com/start

1 July 2014

Alpha Amylase: The previous transformation gave us several hundred colonies. This will make it difficult to find one that has the mutated Alpha Amylase plasmid. We will need to re-transform the PCR product into DH5a. (JB)

7 July 2014

Alpha Amylase: Dr. Grose said that the likely reason for no colonies was due to a diluted amount of PCR product being used since I only used 10 uL of the original mutated PCR product and added water, buffer, and DpnI for the digest I tried. She suggested transforming 10 uL of the PCR product + buffer mix in order to have a better chance of getting colonies with the mutated plasmid. (JB)

Dispersin B: I finished checking the results of my sequencing and to my relief Dispersin B appears to have been successfully transformed in both Colonies A and F. I set up a PCR reaction using the PIG 97 Dispersin Backbone snf the B1 107 and B1 108 Dispersin B primers because my next step is to transform the gene into the pet15b plasmid so I can start testing Dispersin's effectiveness quantitatively against the Biofilm. -JM

8 July 2014

Alpha Amylase: Transformations yesterday do not appear to have worked. Desi said that we could take the previous plate where we got several hundred colonies, pick from single colonies, streak them on a plate to grow them up and make sure we can obtain single colonies since they were so small on the last plate, and run PCR on them to see if there are any that look like they have the plasmid in them. (JB)

9 July 2014

Alpha Amylase: We discovered that the box of DH5a we had been using last week was not actually DH5a as someone had put the wrong tubes in the box. Hopefully this means that the transformations are working (given that we are actually transforming it into DH5a!). The mutated alpha amylase was retransformed into DH5a using 10 uL of PCR product as suggested last time by Dr. Grose since the PCR product was diluted during DpnI digest. (JB)

Dispersin B: Unfortunately after running my PCR product out on gel it doesn't look like I had any product. I had a very small amount of PIG 97 plasmid left so I only used .5 microliters for the PCR reaction. Next week I'm going to take the E. Coli cells I transformed with the PIG 97 Dispersin plasmid on the 20th of May and do a plasmid prep and purification so that I can have more PIG 97 to work with. Instead today I attempted to transform E. Coli cells with the Pet15b backbone so I would have enough to work with. In addition to plating 100 microliters of the transformed cells I diluted some into 5 mL of LB with Ampicillin and 100 microliters into 5 mL of LB with Canamycin in an attempt to grow up liquid cultures. The Canamycin being a negative control. Unfortunately neither the plate nor the cultures showed any growth so the transformation appears to have been unsuccessful. -JM

10 July 2014

Alpha Amylase: Finally, it looks like we may have some progress coming, despite accidentally spilling half of my transformation in the plating process. There was one colony, so it is looking more promising that the digest was effective and we may have one colony that was successfully mutated!

A liquid culture overnight in chloramphenicol was prepared from the single colony and the leftover on the stick was streaked on a chloramphenicol plate. A plasmid prep will be performed on the overnight and this will be sequenced in order to determine if this colony contains the mutant plasmid or the wild-type plasmid. (JB)

AiiA: Today I completed the ligation and transformation of Aii into DH5-alpha. Because my sequencing returned an empty backbone last time, I am hoping that it works this time around. I am planning on picking up the plates tomorrow to prepare for colony PCR. (CZ)

11 July 2014

AiiA: I checked the plates today, and nothing had grown! Because we know it is no longer a problem with the E.coli, I am planning to recheck the digest of the backbone that I did next. (CZ)

Alpha Amylase: Busy day, so I just pelleted the cells down. (JB)

16 July 2014

Alpha Amylase: Today a plasmid prep was performed following the Denville procedure on the mutated alpha amylase colony from last week and this plasmid was then prepped for sequencing tomorrow. Each reaction consists of 2 uL of the mutated plasmid and 1 uL of either the forward signaling sequence alpha amylase primer or the reverse alpha amylase primer. (JB)

Aii: Today we decided with Dr. Grose to redo the amplification of Aii, since we got out a an empty backbone. I used some of the leftover amplified and purified insert that we had obtained previously, and redid the PCR. (CZ)

18 July 2014

Aii: There was a crack in the lid of my PCR tube, so I lost a fair amount of the amplified product. I redid the PCR twice, with one tube using the PCR from Wednesday as template, and another tube with my insert I purified in June.(CZ)

Alpha Amylase: We got back the sequencing results. The PstI restriction site located within the amylase gene remained unchanged meaning the mutagenesis did not work. We will have to redo the mutagenesis procedure on Monday. (JB)

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