Difference between revisions of "Part:BBa K137007:Experience"

Michigan iGEM 2013

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.

Michigan iGEM 2012

When coupled to the constitutive protein generator BBa_K880005, BBa_K137007 produced functional FimE recombinase. Functionality was observed using the asymmetrical digest reporter BBa_K880001.

The asymmetrical digest reporter (BBa_K880001) in the vector pSB3C5 was co-transformed in 10-beta E. coli (NEB) with strong, constitutively expressed FimE (BBa_K880005-BBa_K137007) in the vector pSB1A2. Co-transformants colonies were analyzed for inversion using the asymmetrical digest assay. The asymmetrical digest reporter and fimE (700bp band Fig7, Lanes 2-4) are both present within the co-transformants. The asymmetrical digest reporter was observed in a partially flipped state exhibiting both the 353bp band as well as the 255/238bp bands (Fig 7, Lane 2-4). It has been noted that fim inversions induced by FimE in a previously engineered recombinase systems exhibited low efficiency resulting in a mixed population of flipped and unflipped inversion regions (Ham et al., 2008).

Fig 7. 10-beta E. coli with FimE. Lane 1 is a 100bp Ladder (NEB), lane 2-4 are K880001 co-transformed with FimE showing a mixed state indicated the presence the uninverted K880001 yields a 357bp band, while inversion induced by FimE is expected to result in a 255/238bp band.

See the [http://2012.igem.org/Team:Michigan Michigan iGEM 2012] page for additional details.