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Team:BYU Provo/Notebook/Biofilm/septoct
From 2014.igem.org
BYU 2014 Notebook |
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2 September 2014
Alpha Amylase: Based off the sequencing results from the latest round of colony PCR of alpha amylase, it looks like colony 3 contained a mutated PstI restriction site. From the streak plate and overnight was started. (JB)
3 September 2014
Alpha Amylase: Performed a plasmid prep of the colony 3 mutant following the SpinSmart High-copy plasmid purification. I took the prep and measured the plasmid concentration by nano drop and it only read around 20 ng/uL. This is definitely too low to get any sequencing from. I started a new overnight from the colony 3 plate streak. (JB)
Aii: I obtained colonies on all of the plates that I used to transform the Aii and TolB into E. coli, which I hope is a good sign. My next step in the process is to do colony, which I finished today. I used the plasmid specific primers.(CZ)
4 September 2014
Alpha Amylase: Performed a plasmid prep of the colony 3 mutant again. This time I got a much larger pellet of cells from the overnight. The concentration was around 60 ng/uL however. Not sure why I have been getting such low plasmid concentrations lately. (JB)
5 September 2014
Aii: Today I am running my PCR products from Wednesday out on a standard agarose gel. (CZ)
Alpha Amylase: Today I started four overnights from the alpha amylase mutant colony 3 streak. Hopefully we will be able to obtain a sufficient concentration of the plasmid since the last few rounds of plasmid prep have yielded unusually low concentrations. (JB)
6 September 2014
Alpha Amylase: Today I combined the four overnights down and pelleted them. (JB)
8 September 2014
Alpha Amylase: Today I performed a plasmid prep following the SpinSmart High-copy plasmid purification protocol and materials. Despite the four combined liquid cultures I was only able to get a concentration of 114 ng/uL. This should be sufficient enough though for sequencing. I prepped for sequencing of the mutant colony using the forward and reverse pSB1C3 primers and the new internal primer that was created a few weeks ago. We can also start assaying the amylase in pet15b to determine the pre-mutant efficiency of the protein and later assay the mutant amylase to be sure that the mutation does not decrease the efficacy of the protein. I tested the pH of the biofilm samples to see if we would need to add buffer to them so that the protein would not denature if the pH was hostile. Luckily, the pH range for all of the samples was in the 7.1-7.5 range so we will not need to add any buffer when we start sequencing. (JB)
10 September
Alpha Amylase: Today a majority of the time was spent up in Salt Lake City at the University of Utah retrieving N. multiformis from Ananda Shankar, a graduate student at the university. He was very generous and gave us a culture that he had been growing for the past couple of months, frozen stock, and very specific instructions on how to make the media needed for the N. multiform is to grow.
Sequencing data also came back today for the amylase mutant plasmid prep. (JB)
11 September
Alpha Amylase: Today I spent some more time reviewing the sequencing results. It has been a bit difficult going through and piecing everything together to see if there are or are not any additional mutations to the alpha amylase gene. The coverage was very good and very accurate, but there was a section of the internal primer sequencing that was a bit spotty. There were extra base pairs sometimes, a missing base pair others, or a base pair that could not be read. But the mutation is there! (JB)
12 September 2014
Alpha Amylase: Since there was the spotty section of sequencing where none of the primers picked up the section I prepped two more instances for sequencing, one using the amylase specific forward primer and the other using the internal primer again. (JB)
