Difference between revisions of "Part:BBa K880001"

Revision as of 02:44, 4 October 2012

An Asymmetrically Digestible Reporter Utility for Assaying fim-Based Recombinases

K880001 is a utility for characterizing the functionality of fim based recombinases such as FimB, FimE, and HbiF and consists of K137008 and K137010 flanking a “junk” DNA sequence K165006 containing an off-center AgeI restriction site proximal to the BioBrick prefix; inversion of the fragment will cause the site to move towards the suffix.

Digestion of the unflipped reporter with EcoRI and AgeI will yield a ~70bp fragment when in a FimE-flippable orientation and a ~220bp fragment when in an HbiF flippable orientation. Alternatively, digestion with AgeI and PstI will yield fragments of roughly opposite orientation. The reporter in the registry sequence is FimE flippable.

This is an artificial construct based off the assay performed in Teng et al. Infect. Immun. 2005, 73(5):2923.

Use of this Part in Characterizing the FimE Recombinase

Team Michigan '12 used this part to characterize its recombinase-based switch systems.

To test the hypothesis that the IRL and IRR orientations were the determinative factor to the inversion negative results observed using K880003[1], we created this asymmetrical reporter (K880001). K880001 was engineered such that the IRL is situated on the right of the inverted region and the IRR is situated on the left. This IRL/IRR orientation restores the orientation as found in the natural fimS region (Gally et al., 1996) and a previously engineered recombinase systems (Ham et al., 2006 ).

The new asymmetrical digest reporter (K880001) in the vector pSB3C5 was co-transformed in 10-beta E. coli (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2. Co-transformants colonies were analyzed for inversion using the asymmetrical digest assay</html>[2]. 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 1, 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). To further characterize the new asymmetrical digest reporter (K880001) in the vector pSB3C5 was transformed into E. coli DH5a, a strain with endogenous FimE. In accordance with the co-transformation experiment, the asymmetrical digest reporter was observed in a partially flipped state (Fig 2, Lane 2). [[Image:MichFig7.png|300px|thumb|left|Fig 1. 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. ]] [[Image:MichFig8.jpg|300px|thumb|none|Fig 2. An electrophoresis gel of the asymmetric digest assay of K880001 in E. coli DH5a with endogenous FimE. Lane 1 is a 100bp Ladder (NEB), lane 2 is K880001 showing a mixed state indicated by the presence the uninverted K880001 yielding the 357bp band, and the inverted K880001 resulting in a 255 and 238bp band. ]] Sequence and Features BBa_K880001 SequenceAndFeatures

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 To test the hypothesis that the IRL and IRR orientations were the determinative factor to the inversion negative results observed using K880003[https://parts.igem.org/Part:BBa_K880003], we created this asymmetrical reporter (K880001).  K880001 was engineered such that the IRL is situated on the right of the inverted region and the IRR is situated on the left.  This IRL/IRR orientation restores the orientation as found in the natural <i>fimS</i> region (Gally et al., 1996) and a previously engineered recombinase systems (Ham et al., 2006 ).
-The new asymmetrical digest reporter (K880001) in the vector pSB3C5 was co-transformed in 10-beta <i>E. coli</i> (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2.  Co-transformants colonies were analyzed for inversion using the asymmetrical digest assay (hyperlink to protocol) .  The asymmetrical digest reporter and <i>fimE</i> (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 1, 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).
+The new asymmetrical digest reporter (K880001) in the vector pSB3C5 was co-transformed in 10-beta <i>E. coli</i> (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2.  Co-transformants colonies were analyzed for inversion using the asymmetrical digest assay</html>[https://static.igem.org/mediawiki/2012/a/aa/MichAsm.pdf]<html>.  The asymmetrical digest reporter and <i>fimE</i> (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 1, 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).
 To further characterize the new asymmetrical digest reporter (K880001) in the vector pSB3C5 was transformed into <i>E. coli</i> DH5a, a strain with endogenous FimE.  In accordance with the co-transformation experiment, the asymmetrical digest reporter was observed in a partially flipped state (Fig 2, Lane 2).