Week-by-Week Progress
Week of 9-29-14 through 10- 4-14
This week our team had to split up. Two of our members-Kevin and Lyuda- went along with our adviser Professor Twaddle to Posters on the Hill for CCURI (COmmunity College Undergraduate Research Initiative) in Washingtin D.C. While they were away, Karinne worked on subculturing colonies into fresh broth so that we can make sure to have clean cell lines to work with.
We also continued our collaboration with Carnegie Melon University. They sent us a kit they prepared and we were happy to test it for them. They kit was a fast, easy and simple to understand procedure that visually extracted the DNA from untoasted wheatgerm.
 Broth cultures of top 10, E. coli C, K1477014, K1477030
400 ul of methyl orange used before step 7
Left tube: 50 ul crystal violet dye used before step 7
Right tube: 50 ul crystal violet dye used after step 7
Week of 9-21-14 through 9-28-14
This week we are going to keep working on our assay of Beta-galactosidase activity. So far we have had to unsuccessful attempts at our run-through of it with intact cells. This week we started it a third and fourth time and had success! Our E. coli C turned to a red color along with our K1477014 part. Not only that, but our negative control stayed negative. Everything worked out perfectly except for the time elapsed to get these results. We expected it to take 12-24 hours for a color change to occur, but it ended up taking about 36 hours for E. coli C and a little over 48 hours for the Top 10 in the last attempt. However we are still pleased to have acheived success (we are still fighting contamination.)
We also did two runs of this assay with chemically lysed cells. In the first run through, we got positive results. However, an inquiry led to the discovery that one of the chemicals used could potentially denature any enzymes present, and so we had to throw out that data. The second time we used an approriate chemical, but had positive results on everything-including the negative control. We are now out of our required materials for chemically lysing cells.
Our second successful attempt of our Beta-galactosidase activity assay
Week of 9-14-14 through 9-20-14
This week we began our attempts on the Beta-galactosidase acitvity assay. We plan to run this assay three different ways. We will first complete it using intact cells, measuring the activity that is produce inside the cell. Then we will use chemically lysed cells. By lysing the cells in this way, we are ensuring a higher accuracy in regards to amount of cells being lysed. The chemicals will destroy the cell wall and the beta-gal complimentation can occur in vitro. Finally, we plan to bring the core idea of our research project together, and then we will run this assay using cells that have been lysed with bacteriophage.
In all of these, we will be using chlorophenol red (CPRG) as the substrate. This will cause a color change that ranges from yellow to dark red. We are using Top 10 E. coli cells ad our negative control, as these only produce one out of the two parts that make up the enzyme. We will be using E. coli C as our positive control. We are testing the activity produced in our part K1477014 tranformed into Top 10 cells.
Our first try did not yield correct results, with all three cell samples leading to a positive color change. Our second attempt also did not turn out right. Only the E. coli C gave a positive reading of Beta-gal activity.
In this attempt in our Beta-galactosidase activity assay, our negative control also gave a positive reading.
|
"
