Protein_Domain
hbiF

Part:BBa_K880000:Experience

Designed by: Josh Atkinson, Mike Ferguson, and Ben Parker   Group: iGEM12_Michigan   (2012-09-29)

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Applications of BBa_K880000

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Michigan iGEM 2013

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

Michiganigempartmainfig1.jpg

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.

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