Difference between revisions of "Part:BBa K2324013"

Revision as of 17:57, 1 November 2017

pRha_FimH_1His

This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. The coding sequence is under the control of a rhamnose-inducible promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the fim operon

The part acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of possible E. coli strains (Top10, FimB KO, FimH KO), the fusion protein can be expressed by inducing the culture overnight at 0.6 OD with 2% rhamnose. The production of the 6xHistidine tag can be tested by the use of an SDS-PAGE and a Western blot.

The results of an SDS-PAGE and subsequent Western blot provided bands at the corresponding molecular weights for the FimH_1His protein which also had binding affinities for an anti-His antibody. The result of a band that corresponds to a molecular weight marginally lighter than the entire intact FimH_1His evidentiated expression of the protein, and suggested that the signal peptide had been cleaved upon the proteins delivery to the cell surface membrane. This makes a case for successful export of the protein and therefore successful pilus biogenesis.

Figure 5 The wells of the polyacrylamide gel contained the following, 1-10:marker,ΔFimH_22His pellet, ΔFimB_22His pellet, Top10_22His pellet, ΔFimH_22His pellet, ΔFimH_22His supernatant, Top10_22His supernatant, BL21(DE3)_22His supernatant, ΔFimB_22His supernatant and marker. The band of importance is the one at 28-30kDa which represents the FimH protein, potentially having been exported and had its signal peptide cleaved.

Sequence and Features