Composite

Part:BBa_K2324012

Designed by: Rahan Nazeer   Group: iGEM17_Exeter   (2017-10-16)
Revision as of 19:18, 1 November 2017 by Rahannazeer (Talk | contribs)


pJ23100_FimOp

This part contains the fim operon under control of a constitutive promoter. 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

<figure> <img class="rounded mx-auto d-block w-50" src="T--Exeter--MGNS.jpeg" align="left"> <img class="rounded mx-auto d-block w-50" src="T--Exeter--Top10NS.jpeg" align="right"> <figcaption>Figure 10 On the right is an electron micrograph of an E. coli MG1655 cell. Pili are clearly visible on the surface of the cell. On the left is an image of Top10, which displays no pili. </figcaption> </figure>

<figure> <img class="rounded mx-auto d-block w-50" src="T--Exeter--Top10J23NS.jpeg" align="left"> <img class="rounded mx-auto d-block w-50" src="T--Exeter--Top10AraNS.jpeg" align="right"> <figcaption>Figure 11 Top10 with the fim operon under the control of promoter P_J23100 (left) and P_Ara(right), showed no visible signs of pili expression with insertion of the operon alone. </figcaption> </figure>

<figure> <img class="rounded mx-auto d-block w-50" src="T--Exeter--FimBKOWT.jpeg" align="left"> <img class="rounded mx-auto d-block w-50" src="T--Exeter--FimBKOWTclose.jpeg" align="right"> <figcaption>Figure 12 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 right 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. </figcaption> </figure>

<figure> <img class="rounded mx-auto d-block w-50" src="T--Exeter--FimBKOj23.jpeg" align="left"> <img class="rounded mx-auto d-block w-50" src="T--Exeter--FimBKOPARApili.jpeg" align="right"> <figcaption>Figure 13 We transformed a plasmid containing the fim operon under control of promoters P_J23100(left) 

and P_Ara(right). Both exhibit flagella on their cell surface, but the right electron micrograph shows a suggestion of pili.
This suggests that the P_Ara construct was successful in synthesising pili (minus FimH).</figcaption>

</figure>

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