Once again, the website has undergone a series of changes, such as the new sitemap and the improved background page. A lot of the web pages were polished further, and two new Prezis were created to describe our projects. The team is also working on a logo at this point in time.
The team has finished up the PowerPoint to be presented on the day of the YLC Outreach. In addition, the team made plates as demos for the children using CFP, YFP, GFP and RFP. [Pictures to follow.]
Unfortunately, most of the plates had little to no growth. The vectors used were Kan-resistant while the plates were Amp plates. We discovered that one of the RFP vials was incorrect and not properly bricked. It was only a vector for Kan/Amp resistance and no functioning RFP. So we re-plated that day on Kan plates and got successful growth of the GFP and RFP (from a different vial). We concluded from this that the only successful construct was GFP and that RFP was a self-ligated J23100 promoter vector.
The following day at our meeting with our advisors, we went over the procedures. Andrew discovered that ligase used was 3 years expired so we threw it out and started use of our new ligase from NEB. We were advised to try gel purification after digestion to try to prevent self-ligation. We also feared that the miniprep might have been done poorly for GFP, YFP, and mCherry since they were done by new guys to the task and poorly harvested. So these were grown in liquid culture and re-minipreped.
The promoter was also re-examined, and it was determined that we should switch to J23119. This DNA was rehydrated on the plate and prepared to clone over Friday night.
We plated a Kanamycin resistant synechocystis on plates made with noble agar and glucose, noble agar and no glucose, bacteriological agar and glucose, and bacteriological agar and no glucose.
The transformations from last week seemed to have worked. The CFP is extremely light and looks almost like GFP. We think it may be because our UV light isn't very strong.
The team has continued to attempt to biobrick together the necessary genes that create the carotenoid pathway in E. coli (see last week for more information). To do this, the team has decided to follow the biobrick assembly protocol once more, synthesizing the upstream, downstream and plasmid parts, and then connect them. In order to increase the efficiency and yield of this assembly, gel purification was used to isolate the plasmid for the biobrick assembly. Below is the gel with the upstream, downstream and plasmid parts.
Instead of using the old promoter J23100, the team has decided to instead use part BBa_J23119, the strongest promoter of that constitutive promoter family.
WashUiGEM has continued to develop two different models - a small scale model and a genome-scale model - using MATLAB.
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