Difference between revisions of "Part:BBa K880000"

Revision as of 01:16, 4 October 2012

The HbiF Recombinase

The Hbif recombinase is a putative regulator of the Escherichia coli type 1 fimbriation system, capable of switching the fimS invertible region in the production of fimbriae in a manner similar to the better characterized FimB and FimE recombinases. Orientation control of the fimS switch by recombinases is specific to directionality, with Hbif catalysing the conversion of “OFF” oriented regions to “ON” oriented ones such that the fimS promoter is facing genes responsible for type 1 fimbriae production.

The hbiF gene was synthesized in vitro as a device capable of opposing the directional inversion of the characterized fimE gene K137007 for the purpose of engineering bidirectional molecular switches. A synthetic device using this protein can be made by fusing two unique invertable repeats (IRs) recognized by the recombinase-parts K137008 and K137010, in that order- flanking a sequence of interest of 200-300bp and expressing hbiF or fimE in low-to-moderate levels to cause the sequence to invert:

-FimE will cause a template strand between the IRs to face the upstream coding strand.

-HbiF will cause a coding strand between the IRs to face the downstream template strand.

Note that IRs’ initial orientation is FimE flippable, but not HbiF flippable.

This reaction can be characterized by digesting asymmetrical digest reporter K880001 with EcoRI and AgeI and determining the lengths of the resulting fragments, or by testing for natural E.coli fimbriation, which HbiF activates.

Additionally, HbiF contains an RFC25 suffix and is capable of undergoing protein fusions to fluorescent proteins for quantification, affinity purification tags, or degradation tags such as K880004 to modulate degradation rate.

Reference: Xie et al. Infect. Immun. 2006, 74(7):4039

Characterization: Observation of HbiF induced fimbriation in DH5alpha

Upon transformation of E. coli DH5a with strong, constitutively expressed HbiF (K880005-K880000), we noticed cell aggregates in liquid cultures that were difficult to resuspend upon pelleting. Transmission electron microscopy (TEM) of the culture showed that HbiF expressing cells were fimbriated (Below, A and B), suggesting that HbiF is expressed and actively working on the natural fimS region flipping from “OFF” to “ON” (Xie et al., 2006).

See [http://2012.igem.org/Team:Michigan/Results] for a more complete description of our work involving this part.

TEM micrographs of E. coli DH5a cells transformed with HbiF in pSB1A2 (A, B) versus untransformed E. coli DH5a cells (C, D). Fimbriae are clearly visible in the cells expressing HbiF (A, B), while absent in the control cells (C, D). Bar length indicates 500 nm.

Sequence and Features