Team:WashU StLouis/Broad

Step One: Utilizing E. coli 1303's Ribitol Operon

In order to test for the presence of Puccinia graminis f. sp. tritici (Pgt), we are taking advantage of E. coli 1303’s natural ribitol metabolism pathway. SInce ribitol is unique to Pgt, by detecting ribitol, we will be able to indicate the presence of Pgt. The ribitol operon consists of four separate genes, rbtR, rbtD, rtlK, and rbtT.

rbtD is under constiutive control and catabolizes ribitol into ribulose. This product in turn induces the transcription of ribitol kinase. rtlK is the only gene that requires the presence ribitol in order to be induced. Since we are looking for a system sensitive to ribitol, rtlK was our target for detection. We decided reassemble the ribitol operon, replacing the rtlK gene with a fluorescent protein. With our modifications, the cells will produce a fluorescent signal in response to ribitol.

The ribitol transporter can be found in the registry as the composite part BBa_K2663001.

We have chosen E. coli DH5α to be the chassis for our sensing mechanism because this strain of E. coli are incapable of metabolizing nor naturally interact with ribitol. This eliminates any unwanted activations of the ribitol operon an ensures that any fluorescence produced is due to the presence of Pgt.