Difference between revisions of "Part:BBa K2324013"

Revision as of 19:36, 1 November 2017

pRha_FimH_1His

The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein. 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 (BBa_K902065), with the BBa_B0034 RBS and BBa_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

As well as being utilised as a metal binding protein, this part also acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of E. coli strains BL21(DE3), Top10, FimB KO, FimH KO), the fusion protein can be expressed by inducing the culture at 0.6 OD with 2% rhamnose. The production of the 6xHistidine tag can be probed by the use of an SDS-PAGE and a Western blot.

After 24 h growth, cultures containing the P_Rha_FimH1His and wild-type strains were harvested. Cells were disrupted using BugBuster protein extraction reagent (Merck) and samples of the soluble and insoluble fractions were prepared for SDS-PAGE. Western Blots were probed with an anti-6xHis primary antibody raised in Mouse and anti-Mouse alkaline phosphatase-conjugated secondary antibody and visualised with SigmaFast BCIP/NBT.

Figure 1 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.

Figure 2 The wells is this gel are filled, 0-9, as follows: marker, BL21(DE3)WT pellet,Top10 WT pellet, ΔFimB WT pellet, ΔFimH WT pellet, BL21(DE3) WT supernatant, Top10 WT supernatant, ΔFimB WT supernatant, ΔFimH WT supernatant and BL21(DE3)_22His pellet.

Figure 3 The columns of the Western blot correspond to the following samples,0-9:marker,ΔFimB_1His pellet, Top10_1His pellet,BL21(DE3)_1His pellet,BL21(DE3)_1His pellet,BL21(DE3)_1His supernatant, ΔFimH_1His supernatant, ΔFimB_1His supernatant, Top10_1His supernatant and marker. The bands present here demonstrate the affinity of protein with the same molecular weight as FimH with anti-His antibodies. This would imply that FimH_1His is being expressed.

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.

Sequence and Features