Part:BBa_K895002:Experience

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••• iGEM Dundee 2012

This part was seen work in practice. The protein could be overproduced as an HA-tagged version and verified by SDS-PAGE and Western immunoblotting (Figure 1). In addition,the stability of the protein when used as a fusion was examined. A fusion to mCherry was found to be stable (Figure 2).

Results

Characterisation by Dundee iGEM Team 2012

The ability of the BBa_K895002 biobrick to be stably expressed and produced in the E. coli chassis was examined. An HA-tagged version of BBa_K895002 was cloned into pT7.5 derivative pUNI-PROM (Jack et al. 2004). E. coli strain BL21(DE3) was transformed with the plasmid encoding HA-tagged BBa_K895002 and grown aerobically overnight in LB medium. A 25 ml flask containing LB + 0.5% (v/v) glycerol was then incoculated and grown at 37 C. Following 4 hours growth an aliquot was taken for analyses by SDS-PAGE and Western immunoblotting, and the remainer of the culture was modified by the addition of 1 mM IPTG (final concentration). Following a further 3 hours growth another aliquot of cells were taken for analysis.

The whole cell samples were subjected to SDS-PAGE and Western immunoblotting (Figure 1). A clear IPTG-inducible protein band was observed at the correct molecular mass for VgrG. Western immunoblotting confirmed that the overproduced protein was the HA-tagged VgrG protein.

Figure 1: Production of VgrG in E. coli. Whole cell samples were separated by SDS-PAGE and either stained with Coomassie Blue or blotted onto nitrocellulose for Western immunoblotting. The antibody used was a commercially available mouse monoclonal against the HA epitope.

This small scale assay reveals that VgrG (79.5 kDa) can be stably produced in isolation away from other Type VI Secretion System components in an E. coli chassis. The next issue to address was if VgrG could be engineered as a stable fusion protein. This was done by cloning the BBa_K895002 biobrick as a translation fusion with the fluorescent reporter mCherry. The two proteins were linked using an HA epitope tag, in order to assess whether this was a suitable linker for fusion proteins

Figure 2: Production of Hcp- and VgrG-mCherry chimeras in E. coli. BL21(DE3) was transformed with plasmids encoding either BBa_K895001 fused to mCherry or BBa_K895002 fused to mCherry. Whole cell samples were taken from cultures either with or without 1 mM IPTG in the medium. Whole cell samples were separated by SDS-PAGE and either stained with Coomassie Blue or blotted onto nitrocellulose for Western immunoblotting. The antibody used was a polyclonal against recombinant mCherry.

This small scale assay reveals that large VgrG protein (79.5 kDa) can be stably fused to alien reporter proteins, such as mCherry (115 kDa fusion). This experiment also shows that the HA epitope (YPYDVPDYA) is a stable linker that can be used to build chimeric proteins.

References

The pUNI-PROM version of pT7.5 used to overproduce BBa_K895002 and its derivatives was described in this paper:

Jack,R.L., Buchanan,G., Dubini,A., Hatzixanthis,K., Palmer,T. & Sargent,F. (2004) Co-ordinating assembly and export of complex bacterial proteins. EMBO J. 23:3962-3972.

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