Part:BBa_K2100014:Experience

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Applications of BBa_K2100014

Using the mCherry entry vector, the MIT iGEM team constructed the pDEST-mCherry through polymerase chain reactions. The plasmid was used as a destination vector in many of our LR reactions during the cloning process. Once we transformed the product of the LR's into E. Coli and plated the transformed bacteria onto Agar plates, we noticed pink/red and white colonies once they grew up. The pink/red colonies are the ones with plasmids that did not lose the mCherry gene during the LR reaction, meaning that those are still destination vectors. The white colonies are expression vectors, because the lack of pink indicates the LR reaction worked and the mCherry gene was replaced by the gene and promoter of interest. The following image of a plate shows the two types of colonies (pink and white) grown up from an LR reaction using pDEST-mCherry GG as a destination vector.

Our team used trial and error to conclude the optimal concentration for plating transformed LR's completed with the pDEST-mCherry GG. We found plating 5 uL of the transformation with 200 uL of water gave the best results for ease of picking colonies. We also found that over time, as the colonies grow, the red color becomes more pronounced. When it is not, and a colony with mCherry mistakenly gets picked, after centrifugation the pelleted cells will be a light pink.

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