Part:BBa_K2324012

pJ23100_FimOp

This part contains the fim operon , minus the fimH under control of a constitutive promoter. The constituent genes code for structural proteins, such as FimA which makes up the majority of the main body of a pilus, and others which support the process of pilus biogenesis. When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The part requires no induction, and therefore is able to continuously produce the large mass of protein encoded in the operon.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2648
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 931
  Illegal AgeI site found at 962
  
• 1000
  COMPATIBLE WITH RFC[1000]

Fim Operon expression

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<figcaption style="text-align:center">
 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.
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</figure>

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).