Part:BBa_K2324011:Design
T7_FimH_225sfGFP
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 451
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This part was used for initial expression studies. The sfGFP protein was inserted into the amino acid sequence at residue 225 and the whole coding sequence was under control of the strong, well characterised T7 promoter. Use of this promoter in conjunction with the strong, well characterised B0034 RBS and B0015 terminator was intended to limit the degree to which the sub-parts, other than the CDS, would contribute to the overall unpredictability of the system.
The intended purpose of the sfGFP was to act as a reporter. As a large protein, it would be the most likely to affect pilus biogenesis, especially the export process. The fluorescent property would give an unambiguous answer to the question of whether the FimH fusion protein could fold and whether pilus biogenesis could occur.
Our experimental approach was therefore to transform the plasmid into BL21(DE3) and to grow a culture of a consequent colony. This culture was grown to an OD of 0.6 with a 50 ml volume at which point it was induced with rhamnose solution, up to a 2% concentration. The induced culture was left overnight to allow for protein expression and export before being examined using a plate reader and a flow cytometer.
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Figure: Shown here is a graph of OD adjusted fluorescence. It demonstrates the difference in fluorescence between the wild type BL21(DE3) and BL21(DE3) transformed with the T7_FimH_225_sfGFP construct. There is a signficant difference in the absorption at the given wavelength, which suggests successful expression of the FimH_sfGFP fusion protein.
These results demonstrate a distinct difference in fluorescence between our modified bacteria and the wild type controls. The image stream data show a proportion of the present cells in the culture to exhibit significant fluorescence. Consequently, the conclusion can be drawn that the pili containing FimH fused with sfGFP were successfully produced by the cell. This is evidence that FimH can be exported from the cell, even with a very large protein domain fused to it, and so it stands to reason that smaller domain fusions should cause no issues. This makes a strong case for the ongoing develop of a modular toolkit of substrate-binding pili.
Design Notes
Codon optimisation for expression in E. coli . Research was required to identify the ideal placement of the foreign protein domain.
Source
The CDS is the new part BBa_K2324001.The RBS and terminator are the registry parts BBa_B0034 and BBa_B0015 respectively. The T7 promoter is part BBa_K1614000.