Part:BBa_K2324015

Arabinose inducible fim operon

This part contains the fim operon under the control of an inducible arabinose promoter (BBa_I13453). The operon contains six Fim proteins, including FimA, FimI, FimC, FimD, FimF and FimG (Le Tong 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 (, however we used the modular cloning strategy to construct our complete operon from three sections (https://parts.igem.org/Part:BBa_K2324016, https://parts.igem.org/Part:BBa_K2324017 and https://parts.igem.org/Part:BBa_K2324018), which has produced scar-sites between the three sections. We also codon optimised the sequences using the IDT E. coli codon optimisation software.

Fim Operon expression

Figure 1: Top is an electron micrograph of an E. coli MG1655 cell. Pili are clearly visible on the surface of the cell. Bottom is an image of Top10, which displays no pili.

Figure 2 : Top10 with the fim operon under the control of promoter P_J23100 (top) and P_Ara(bottom), showed no visible signs of pili expression with insertion of the operon alone.

Figure 3: ΔFimB should not, theoretically, produce pili. The regulatory gene FimB has been knocked out, and so the operon has effectively been switched off. These electron micrographs show wild type ΔFimB with strong, peritrichous flagellar expression, but no visible signs of pili. The image on the bottom shows little evidence of pili or flagella connected to the cell surface, which suggests that the negative staining technique can be damaging to these structures and could cause detachment and fragmentation.

Figure 4: We transformed a plasmid containing the fim operon under control of promoters P_J23100(top) and P_Ara(bottom) in ΔFimB. Both exhibit flagella on their cell surface, but the bottom electron micrograph shows a suggestion of pili. This suggests that the P_Ara construct was successful in synthesising pili (minus FimH).

Conclusion

The electron micrographs presented above 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 investigate further, particularly in combination with out modified FimH constructs.

References

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  Illegal BamHI site found at 2743
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1026
  Illegal AgeI site found at 1057
  
• 1000
  COMPATIBLE WITH RFC[1000]