Arabinose inducible fim operon
This part contains the fim operon under the control of an inducible arabinose promoter (BBa_I13453). The operon consists of coding sequences for expression of six Fim proteins, including FimA, FimI, FimC, FimD, FimF and FimG (Le Trong et al 2010). When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The production of the operon must be initiated by inducing the culture at 0.6 OD and 2% arabinose.
Previous iGEM team, Harvard 2015 synthesised a very similar part (http://parts.igem.org/wiki/index.php?title=Part:BBa_K1850013), however we used the modular cloning strategy to construct our complete operon from three basic parts (http://parts.igem.org/Part:BBa_K2324016, http://parts.igem.org/Part:BBa_K2324017 and http://parts.igem.org/Part:BBa_K2324018), which has resulted in scar-sequences between the parts. We also codon optimised the sequences using the IDT E. coli codon optimisation software.
Fim Operon expression
The electron micrographs presented above of, MG1655 and ΔFimB carrying the fim operon under control of the arabinose inducible promoter, are reminiscent of those produced by Pallesen et. al. and suggest that under control of the arabinose inducible promoter, the fim operon is expressed and pili (albeit incomplete as they are lacking FimH) are formed on the cell surface. Further work is needed to fully characterise this, particularly in combination with out modified FimH constructs. We have successfully shown a suggestion of pili using the arabinose inducible promoter and no pili production using a constitutive promoter.
Le Trong, I., Aprikian, P., Kidd, B. A., Thomas, W. E., Sokurenko, E. V., and Stenkamp, R. E. (2010) Donor strand exchange and conformational changes during E. coli fimbrial formation. Journal of Structural Biology 172, 380–388.
PALLESEN, L., POULSEN, L. K., CHRISTIANSEN, G., and KLEMM, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 125
- 21Illegal BamHI site found at 65
Illegal BamHI site found at 2743
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 1026
Illegal AgeI site found at 1057
- 1000COMPATIBLE WITH RFC