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UNIQa4cfe9a3529c5d2c-partinfo-00000000-QINU UNIQa4cfe9a3529c5d2c-partinfo-00000001-QINU

12/04/2015

To test the presence of ProEP-B2 in the cell, we initially performed a 60 minute fluorescence assay of ProEP-B2 transformed E.Coli media containing substrate quencher 5-FAM at various levels of rhamnose and glucose, at 455 excitation and 515 emission (graphs below). The ProEP-B2 vector is rhamnose inducible and glucose repressible, so we expected that the fluorescence would show discernible increase with increasing rhamnose over time and a decrease with increasing glucose. However, we could not come to a conclusion on whether or not the protein was present in this assay due to the fluorescence of the 5-FAM in the media showing inconclusive patterns in the presence of both the rhamnose and glucose.

Because the media assay was unsuccessful, we then performed another 60 minute fluorescence assay using ProEP-B2 transformed E.Coli cells lysed with SDS lysis buffer, again containing 5-FAM at various concentration of rhamnose and glucose at 455 excitation and 515 emission. This assay successfully showed that the protein was present within the cells due to increase in fluorescence over time. However, like with the previously performed media assay, the growth of the cells was not affected by increasing rhamnose and glucose concentrations. This may have been due to the samples containing a much higher concentration of cells compared to glucose so glucose inhibition was indiscernible. Another explanation is that there was too great of a ProEP-B2 protein concentration within the sample before the assay was conducted, and so the glucose and rhamnose had little observed affect.

Final Conclusions: We cannot conclude that the Yeb F secretion vector was working properly due to the little effect that rhamnose and glucose had on the vector, but we can confirm that the ProEP-B2 was present in the cells.