14 February 2014

19 February 2014

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)

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)

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)

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)

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)

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)

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