Composite

Part:BBa_K2324015

Designed by: Karolina Kostrzynska   Group: iGEM17_Exeter   (2017-11-01)
Revision as of 22:49, 1 November 2017 by Rahannazeer (Talk | contribs)


Arabinose inducible fim operon

This part contains the fim operon under the control of an inducible arabinose promoter. The operon contains six Fim proteins, including FimA, FimI, FimC, FimD, FimF and FimG. 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 overnight at 0.6 OD at 2% arabinose.

Previous iGEM team, Harvard 2015 has synthesised a very similar part, however we used the modular cloning strategy, which has produced scar-sites at various sequences and it has also been codon optimised by the IDT E. coli codon optimisation software.

Fim Operon expression

T--Exeter--MGNS.jpeg

T--Exeter--Top10NS.jpeg

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.


T--Exeter--Top10J23NS.jpeg

T--Exeter--Top10AraNS.jpeg

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.

T--Exeter--FimBKOWT.jpeg

T--Exeter--FimBKOWTclose.jpeg

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.

T--Exeter--FimBKOj23.jpeg

T--Exeter--FimBKOPARApili.jpeg

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).


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]


[edit]
Categories
Parameters
None