Difference between revisions of "Part:BBa K880000"

Latest revision as of 16:36, 29 September 2013

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

The 2013 Michigan igem team successfully used this part to completely flip the fim switch in the desired direction.

Fig. 1 - The fim transcriptor is capable of changing states completely and unidirectionally

NEB 10-beta E. coli, which lack the native fim switch (fimS) and known fim recombinases, were co-transformed with either constitutive hbiF or fimE and BBa_K1077007 (J23100 fim switch, ON orientation), plated on LB plates and grown overnight for ~16 hours. The state of the switch was assayed by using an asymmetric digest assay on PCR amplified switch. There are two hincII sites located within the K1077007 switch, one of which changes position depending on the state of the switch. The result is that when the switch is in the ON position, a 870bp, 273bp, and 248bp band is produced. When the switch is in the OFF position, a 680bp, 473bp, and 273bp band is produced. A and B)The digest assay was quantified using densitometry and showed greater than 95% of the switch in the desired state (ON when co transformed with constitutive hbiF and OFF when co transformed with constitutive fimE). This is consistent with hbiF’s previously observed functionality of catalyzing the inversion of fimS from the OFF to ON orientation and fime’s previously observed functionality of catalyzing the inversion of fimS from the ON to OFF orientation. C and D) A gel of the digest assay fragments quantified in A and B.

The faint ~870bp band in 1D, seemingly indicating residual OFF state, may correspond to the constitutive recombinase generator which is 858bp. We did not have time to cure the bacteria of the constitutive recombinase generator plasmid. Additionally, the switch is on the high copy pSB1C3 plasmid and so it could be that some switch plasmids are escaping recombination. We did not have time to move the switch to a low copy plasmid or the chromosome.

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