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BYU 2014 Notebook Edit February April

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14 February 2014

We worked on some preliminary research regarding the biofilm aspect of our project idea. A good potential gene to look into is DispersinB which last year's BYU iGem team worked with some, so we can talk to team members from last year regarding that. Below are links for papers worth looking into more. (JB)

http://www.ncbi.nlm.nih.gov/pubmed/23103508

http://www.uniprot.org/citations/12896987

http://www.nlm.nih.gov/medlineplus/ency/article/000679.htm

19 February 2014

Today we read some more on the biofilm being formed in activated sludge processors like the one in Park City, UT. We are searching for different enzymes that could be utilized to break down the biofilm buildup found in these types of places. (JB)

11 March 2014

We emailed BYU's iGem team from last year which had done some work on biofilm degradation with alpha amylase and dispersin B. They gave us some good papers to look into regarding this aspect of our project. Another gene we have been looking into is Aiia, which is a quorum sensing blocker, which would be helpful in preventing aggregation of bacteria into biofilms. (JB)

17 March 2014

Today we went over our respective presentations for our subprojects. We will need to be able to insert our modified plasmid with our biofilm inhibiting genes into the N. multiformis bacteria which will then produce the enzymes and quorum sensing components and will affect the other bacteria forming the biofilms in the ASPs. We will need to look at fitness costs later on down the road and select for the most fit mutants with our biobrick in it. (JB)

19 March 2014

Today we started doing research as to how we will insert our genes of interest into the plasmid. Hopefully our genes will be compatible with the standard iGem E. coli plasmid pSB1C3. We need to find the gene sequences that will be used and design primers with restriction sites on them so that we can amplify the gene and insert it into the plasmid. We will also need to start thinking of experimental methods of how to test the efficacy of the different genes in biofilm dispersal and inhibition. I will be doing the research for alpha amylase.

BYU 2013 iGem team’s alpha amylase part: BBa_K1195001

Uniprot: I6S010 (E. coli alpha amylase)

As E. coli is a gammaproteobacteria and N. multiformis is in the betaproteobacteria class, if there are issues that arise from the E. coli gene being incompatible with the N. multiform is, we will need to consider using the betaproteobacteria Alpha Amylase found on Uniprot (Uniprot:Q47IJ1), but this would require isolating this gene ourselves from a betaproteobacterium. Alpha Amylase requires calcium for activation. We will need to verify its presence in the ASPs. (JB)

21 March 2014

We discussed as a group a game plan for our next presentation. We know that the alpha amylase from E. coli that is in the iGem registry will be compatible with the pSB1C3 plasmid. We will need to discuss further how to link all 3 genes together and we will also need to determine if the genes will need to go on the plasmid in a certain order or if they will need anything additional in order to complete their function. We also discussed some preliminary ideas as to how we will test the efficacy of the biofilm inhibition by our components and also the concern whether or not they will be effective at degrading existing biofilms.

I began designing my primers with restriction sites today for the Alpha Amylase gene that is from the BYU iGem registry. The restriction sites used will also depend on the order of linkage of the other genes that will go in our plasmid. (JB)

24 March 2014

Alpha Amylase: Today I continued to work on designing the primers for Alpha Amylase. They are all done except for the promoter/Shine Dalgarno sequence. Dr. Grose showed us how to search for signaling sequences. (JB)

26 March 2014

Alpha Amylase: Today we finished preparing our presentation that we are giving later today. I finished the primer designs but there were a lot of potential hairpins and self-annealing sites that we will have to take a closer look at. As well, the PstI restriction site is contained with the alpha-amylase so we will not be able to use that restriction enzyme with Alpha Amylase until we remove the site with site-directed mutagenesis. (JB)

27 March 2014

Alpha Amylase: Today I did some research into methods other scientists have employed for measuring biofilm dispersal and degradation. (JB)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932551/pdf/1249-06.pdf

http://download.springer.com/static/pdf/846/art%253A10.1007%252Fs12010-011-9526-2.pdf?auth66=1396038196_95251369f639464eb174e81ed448c024&ext=.pdf

28 March 2014

Alpha Amylase: Today we talked to Dr. Grose about designing our primers. I also looked into oligocalc to see if my primers for a-amylase will be viable. The forward primer is fine, the reverse primer needs more reverse complementary base pairs added to the beginning to increase the melting temperature. Dr. Grose also told us to only use the Xba portion of the prefix to cut down on primer length and only use SpiI so that we can clone a-amylase without the PstI restriction site for initial cloning of the gene (we have to do this because a PstI restriction site is found in the middle of the a-amylase gene). (JB)

31 March 2014

Alpha Amylase: Today we learned about site-directed mutagenesis

2 April 2014

Alpha Amylase: Today I continued to work on the procedure write-up for Alpha Amylase. (JB)

4 April 2014

Today I continued to work on fine-tuning the protocol for a-Amylase. Received some clarification on the steps needed in order to both insert the signaling sequence into the gene as well as use site-directed mutagenesis to remove the PstI restriction site from the a-Amylase gene from BYU. (JB)

7 April 2014

Today we spent the majority of the time working on compiling the different aspects of our protocols paper as a group.

9 April 2014

Alpha Amylase: I found a possible contact for obtaining a Nocardia sample in case we want to test the biofilm degradation since Nocardia is a large component of the biofilm found in Activated Sludge Processors. (JB)

We also transformed several of the iGem promoters into E. coli. This was done in order to test the efficacy of the different promoters so that we can known which ones are the best to use for our project. We followed the cloning procedure found in the iGem protocols packet Dr. Grose gave us at the beginning of the semester.

11 April 2014

Today we performed a plasmid prep on the plasmids containing the promoters that we were working with the other day. We followed the Denville Plasmid Prep protocol #5 (SpinSmart Plasmid Purification Protocol: High-copy plasmid DNA from E. coli). (JB)

14 April 2014

Today we compiled the information that we have gathered for our final protocol and started planning our powerpoint for the presentation on Monday we will be giving to our team.

21 April 2014

Today we presented our biofilm degradation presentation to the team.

29 April 2014

Today we worked on the transformation of the plasmids containing the different promoters into DH5α.

For alpha-amylase we are testing both PIG92 and PIG98 to see if one or both of them are the alpha amylase and then we will transform whichever works. Tomorrow after PCR is done we will run 5 uL in gel. Along with the two potential alpha amylase plasmids we also performed PCR on the DispersinB plasmid in the iGem catalog and we performed a transformation of Aiia into DH5α.

1 May 2014

Aiia: Today a plasmid prep of the Aiia plasmid was performed following the Denville SpinSmart Plasmid Purification protocol.

Alpha Amylase: Today sewing PCR was performed on the alpha amylase forward primer pieces. In order to PCR with the overlap extension primers that contain our signal sequence we will need to PCR with just the forward and reverse with the signal sequence and no template so that we have a combined forward primer to use. Then we will use that combined primer as a forward primer in a normal Q5 PCR reaction with the template. The only difference is that 2 uL of each primer should be used (the forward signaling sequence primer and the reverse signaling sequence primer). (JB)

2 May 2014

Today we ran our sewing PCR products on agarose gel and each got a product around 150 base pairs as it should have if the reaction worked properly. We then set up the next reaction with the newly sewn forward primers with the signaling sequence and the reverse primers for each gene as well as the template. We have a 5 uL control for each and then a reaction for each of the parts (including the two Amylase parts we are testing). The tube labeled Amy1 is pIG 92 and the tube labeled Amy2 is pIG98.

5 May 2014

Today we ran the gel of our PCR products of the enzyme plasmids with our sewn forward and regular reverse primers.

Aiia had a product that was around 2500 bp, where is should have only been around 800 so we need to figure out if it amplified the whole plasmid.

DispB showed smearing so either we need to play with the annealing temperatures because the primers are either not attaching at this temperature we used or the plasmid needs to be purified more.

Amy2 was the best band. This was from PIG98. We will need to digest this and the backbone and run it on low melt gel.