Team:Rock Ridge Virginia/Results

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Project Results

Parts

Once we received our parts from IDT we transformed the plasmids in DH5-alpha cells in ampicillin resistant plates.All of our plates (OspA, WSP and GFP) had many colonies due to the fact that we added 3 times the amount of DNA since we had some problems with transformation in our practice rounds. The positive plates were positive and the negative plates were negative.

We picked 5 colonies from each plate and we purified the plasmids using mini-preps. We checked the plasmid purity using agarose gels, all of the colonies had the plamid inside. We did not sent the plasmids for sequencing due to the cost, we just selected one of the five colonies and did a PCR using Gibson Assembly primers. We had a lot of difficulty doing the PCRs because the melting temperature between the primers was not similar. It took us quite some time to get all of amplicons ready, especially WSP to which we had to add b-mercaptoethol to get a good yield.

Our first attempt at PCR

Optimized PCR amplicons :)

For the Gibson Assembly we used a linear ampicillin plasmid backbone (pSB1A3), and the gel purified amplicons for the reaction. Due to the way we designed our primers and parts (composites), we could only do the Gibson Assembly between GFP and WSP. RFP cells are present in the plate and at first we thought it was contamination until we read that it was undigested plasmids.

Problems

Our main problems were of human error, whether not labeling something properly or leaving an experiment half way because a team member had to go to work or practice.Our team was mainly composed of Freshman (who can't drive) and it was difficult for some of them to attend our meetings.

Our next problem was equipment, being from high school it was really hard to maintain our competent cells in dry ice.

We are not sure if the fluoresece of the GFP is real, since it is faint, we are seeing some teams doing exposures of 30 seconds so we might do the same, but this may not be helpful for a scientist on the field. Therefore we also wan to try RFP and see which will give us the best results or fluorescence.

Towards the end of the summer we had to abandon our project to do more fundraising for the upcoming Giant Jamboree. As soon as school started it was very difficult for us to meet and continue our work.

Another problem is the size of our parts OspA is 993bp, WSP is 885 and GFP is 870. This means that characterizing these proteins will also be difficult since they will have a similar molecular weight. There is no antibody for WSP so that means we'll have to have it made before we can characterize it.

Even though we had many difficulties and next year it will still be an uphill battle, although, not as big as the one this year. We are more excited than ever about our project, we think it holds true potential and we might be able to make a difference in peoples'lives.

Our goal is to make a company and develop more living vaccines for other insect born diseases such as: malaria, West Nile virus, Chagas etc.