Part:BBa_K137058:Experience

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• Michigan iGEM 2012

Negative Fluorescent Reporter (BBa_K137058) Data To initially test the FimE recombinase, we utilized a reporter created by the CalTech ‘08 iGEM team (BBa_K137058) for the same purpose. The BBa_K137058 reporter 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. Despite several co-transformation trials, no fluorescence was observed in contrast to CalTech ‘08’s results. Negative Asymmetrically Digestible Reporter (BBa_K880003) Data An asymmetrical digest reporter (K880003) in the vector pSB3C5 based on the inversion region found in K137058 was co-transformed in 10-beta E. coli (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2. Colonies as well as broth subcultures of co-transformants were analyzed for inversion using the asymmetrical digest assay (hyperlink to protocol) . The asymmetrical digest reporter and fimE are present within the co-transformants; however, the asymmetrical digest reporter remains in the uninverted state (Fig 5, lane 3). Fig 5. A representative electrophoresis gel of the asymmetric digest assay of K880003 co-transformed with FimE. Lane 1 is a 100bp Ladder (NEB), lane 2 is K880003 transformed alone, lane 3 is K88003 co-transformed with FimE, and lane 4 is FimE transformed alone. Uninverted K880003 yields a 357bp band as seen in lane 2 and 3, while inversion induced by FimE is expected to result in a 255 and 238bp band. Observation of IRR and IRL Orientation: Scrutiny of the DNA sequence provided a possible explanation of the negative results. As constructed, verified by sequencing, the orientations of IRL(BBa_K137010) and IRR (BBa_K137008) within the inversion region of BBa_K137058 were orientated 180º when compared to the natural fimS region (Gally et al., 1996) and a previously engineered recombinase systems (Ham et al., 2006). This resulted in the IRL and IRR being oriented such that the external half-sites are localized adjacent to the inversion region, while the internal half-sites are located externally (Fig 6). We hypothesize that this 180º orientation of the IRL and IRR is the determinative factor to the inversion negative results. Fig 6. Comparison of the natural fimS IRR and IRL orientation to that of K137058. The orientation of IRL (Blue-Yellow-Orange) and IRR (Purple-Yellow-Green) are both exhibiting 180º orientations in K137058 (B) when compared to the natural fimS region (A). Modified Asymmetrically Digestible Reporter (BBa_K880001) Data: To test the hypothesis that the IRL and IRR orientations were the determinative factor to the inversion negative results observed, we created a second 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 (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 (hyperlink to protocol) . 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. Negative Modified Fluorescent Reporter (BBa_K880002) Data: To additionally test our hypothesis we made a new version of the fluorescent reporter BBa_K137058 with the IRR and IRL orientation, analogous to K880001. Unexpectedly, we did not observe fluorescence possibly attributable to partially flipped states observed in the K880001 co-transformation assays (Fig 7 above) See the [http://2012.igem.org/Team:Michigan Michigan iGEM 2012] page for additional details.

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