Team:Georgia State/Interlab

Team:Georgia State - 2016.igem.org

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Team: Georgia State IGEM

INTERLAB


Our Interlab study was conducted using a BD FACSCanto II RUO flow cytometer. The test devices and controls were transformed into NEB DH5alpha competent cells. Next, picked colonies were grown in Luria Broth overnight and converted to glycerol stocks for -80˚C storage. Then, 2 sets of overnight cultures were grown in Luria Broth (Trial 1) and Terrific Broth (Trial 2). Initial culture OD600 measurements were taken and then diluted accordingly. Next, both culture sets were grown in a shaker/incubator for approximately 6.0 hours at 37.0˚C 250 RPM.

Once finished incubating, the culture sets were prepared for measurement. First, the flow cytometer was calibrated with Sphero Rainbow Beads. Next, the positive and negative controls for each trial were measured to configure FIT-C population gating. After that, each trial was measured individually until 10,000 events were reached. Finally, all trial measurements were batch analyzed and compared to see if media content affected the amount of recombinant GFP expressed.

After analysis of both trials, the 3rd replicates of test device 3 yielded the highest amount of GFP expression. The 3rd replicate in the Terrific Broth trial expressed approximately 3000 MEFL higher than the Luria Broth trial. This was most likely due to a higher abundancy of carbon sources available in Terrific both which support prolific growth conditions in Escherichia coli. Finally, by comparing the amount of GFP expressed in test devices 1, 2, and 3 it was determined that the promoter strengths were ranked low, medium, and high respectively.



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