Results

Liquid cultures of our bacteria were prepared in King’s Medium B supplemented with tryptophan. The culture was centrifuged after growth and the supernatant was filtered and used in HPLC. The figure shows tryptophol run in the same media as our sample. Our HPLC graphs do not have the peak at 162 m/z shown in the standard.

From the HPLC results, the modified bacteria do not show production of tryptophol. One potential explanation is that there is post-translational modification of these gene products. In particular, ARO10 has been shown to be expressed without exhibiting 2-oxo acid decarboxylase activity, indicating the presence of post-translational modification that may be present in yeast cells but absent in the modified E. coli cells.

Most of our time over the summer was spent getting all three genes into one cell. In future experiments, PDC 1 could be used in place of ARO10 to produce the decarboxylase enzyme necessary for the degradation of tryptophan to tryptophol.