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.
Week of 9-7-14 through 9-13-14
Continuing in our goal towards collaboration with other iGEM teams, we came into contact with the Cornell iGEM team. They are working on detecting heavy metal pollution in water. To aid them in their research, we sent them two 50cc water samples from local sources (including the Saint Joseph river and Potato Creek.) Hopefully this helped them out!
As well as our journey towards working with other teams, this week we reverted back to running an electrophoresis gel for our parts. With the help of a new addition to our advisers-Shaunasee Kocen-we found newer materials, and began using different sized DNA ladders to compare. After a few more tries, we were able to get our DNA bands looking great, and we are ready to send our parts into the iGEM registry.
K1477014
K1477030
Week of 8-31-14 through 9-6-14
The South Bend Ivy Tech team has decided that we are going for the gold medal at the Giant Jamboree. This week we began collaborating with other iGEM teams to help them progress in their own research as well as help ourselves reach a new level of accomplishment. To begin, we exchanged emails with the ETH Zurich team. They asked us to complete surveys as well as send them to friends and family members. Altogether, we were able to fill out over 30 surveys. However, we were not able to reach our goal of 50 which was disappointing. Still, the Zurich team graciaously rewarded our efforts with a team badge for our wiki in appreciation of completing the surveys.
Week of 8-24-14 through 8-30-14
This week our team's persistance and effort came to fruition when we finally acheived a virus titer plaque assay that turned out perfectly! There was no contamination. The cell growth did not overpower the working virus bacteriophage. Best of all, we have clear dead zonces that show consistancy with the corresponding viral dilutions. We are now ready to begin performing the Beta-galactosidase activity assay.
Top 10 grown at an initial Absorbancy (A600) of 0.200 with and added T7 bacteriophage viral dilution of 10^-3
Top 10 grown at an initial Absorbancy (A600) of 0.200 with and added T7 bacteriophage viral dilution of 10^-4
Top 10 grown at an initial Absorbancy (A600) of 0.200 with and added T7 bacteriophage viral dilution of 10^-5
Week of 8-17-14 through 8-23-14
We tried again to get good results of the virus titer plaque assay. We are still getting a bit of contamination, although the growth is not bright yellow as the rest of the contamination has been. This leads us to believe that we are still using a concentration of cells that is too high. We have been using Top 10 E. coli cells with an A600 reading of ~0.400. We are now going to start using a broth culture of cells with an A600 reading of ~0.200.
DH5alpha grown on a LB agar plate with an added T7 bacteriophage viral dilution of 10^-8
DH5alpha grown on a LB agar plate with an added T7 bacteriophage viral dilution of 10^-9
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