Part:BBa_K1323014:Experience

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Applications of BBa_K1323014

To characterize this variant of the xylose inducible promoter, it was cloned in front of GFP so a simple fluorescence assay could be performed. The new mutated xylose promoter should be activated in the presence of media containing 2% xylose, assuming its function is no different than the original [1]. The positive control was a plasmid containing the constitutive J23101 promoter in front of GFP. The negative control in this assay was using a plasmid including GFP with no promoter. All plasmids were transformed into the DH5alpha Escherichia coli strain.

Strains with the plasmids were grown to an OD600=0.2 and then were diluted into media and put into a plate reader. Measurements of OD600 and GFP expression were taken at intervals of 10 minutes until the positive control expressed the GFP promoter.

The OD600 graph shows that all of the strains grew in the 96-well plate. The GFP fluorescence intensity graph shows that the J23101 promoter effectively expressed the GFP protein while the strain with no promoter did not have GFP expression. When the xylose inducible promoter was in just LB media, it did not express GFP. When xylose was added, the promoter also did not express GFP. This means that the mutation made to remove the illegal site also affected the promoter. The mutated promoter does not work, even when it is induced with 2% xylose.

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