Team:WashU/Week2

Week 2

YLC Collaboration: After checking on an overnight culture of the transformed cells with the BioBricks from Week 1 on ampicillin plates, we noticed that the eYFP and eCFP bacteria were not transformed well (The plates had low numbers of colonies). Thus, we performed five transformations. We redid the faulty eYFP and eCFP transformations, added two other YFP plasmids and also added a new biobrick, mCherry, proven to exhibit a more vibrant red color than our original RFP.

Five biobricks used for transformations this week:

6. YFP: [http://partsregistry.org/Part:BBa_I13003:Design BBa_I13003]
7. YFP 2: [http://partsregistry.org/Part:BBa_I13002:Design BBa_I13002]
8. mCherry: [http://partsregistry.org/Part:BBa_J06702:Design BBa_J06702]
1. Old eYFP[http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%3Ftitle%3DPart%3ABBa_E0430&sa=D&sntz=1&usg=AFQjCNGQMpvic9Hi4iih_Ayd4jQd0fDmSQ BBa_E0430]
4. Old eCFP: [http://www.google.com/url?q=http%3A%2F%2Fpartsregistry.org%2Fwiki%2Findex.php%3Ftitle%3DPart%3ABBa_E0420&sa=D&sntz=1&usg=AFQjCNGqh4nrV86rOPlesY61Pf7iLz8FNA BBa_E0420]

Afterwards, we grew the cells up on plates with ampicillin, picked colonies and prepared liquid cultures. Then, we prepared minipreps for all of the transformed cells and ran a gel. The results are shown below.

Agarose gel of DNA to make zeaxanthin in E. coli (Brian and Lucas's gel)

Agarose gel of DNA to make fluorescent proteins ("the gel master's" gel)

Website work: Each team member completed his biography, after which the webmaster uploaded them to the website. Work was also done on other parts of the website, including the header, safety page, sponsor section, and media page. The team plans to post pictures on the website detailing all of the misadventures of the iGEM 2012 team, and may even delve into film, should time permit. Finally, plans are in the air for a potential new logo and new banner for the website.

Saffron in a Kan: Synechocystis: The product of days of effort and multiple revisions, the polycistronic construct for the main project finally has been completed and submitted. The order has been sent to DNA 2.0, and we expect our gene to arrive within the next few weeks.

Saffron in a Kan: E. coli: We plan to create a strain of E. coli that will produce zeaxanthin. This will allow us to test if our gene will be able to produce its products in both synechocystis and E. coli. Although the gene has been optimized for synechocystis, it will still be able to make the necessary products in E. coli. More biobricks were chosen to use for this part of the project, including [http://partsregistry.org/Part:BBa_K152005:Design BBa_K152005] (a synthetic pathway for E. coli to produce beta carotene) and [http://partsregistry.org/Part:BBa_I742158:Design BBa_I742158] (CrtZ for E. coli).

We transformed E. coli cells with these plasmids and grew them up on plates with ampicillin. Then, we picked a colony from each plate (2 total one for each plasmid) and grew the bacteria in liquid culture. In the end, these colonies did not produce the results that we wanted. A second attempt will be made next week.