Difference between revisions of "Part:BBa K880002"

Latest revision as of 01:47, 4 October 2012

A Fluorescent Reporter for Assaying fim-Based Recombinase Activity

-IRR K137008 + inverted tetR promoter K137047 + IRL K137010 + GFP I13504 (MI275) -GFP fluorescence reporter to assay the activity of FimE K137007 and HbiF K880000 recombinases.

This has the same components as K137058 with the inverted repeats reversed such that the external half sites are external to the flip region (IRR - “flip region” - IRL)

It is a GFP fluorescent reporter capable of assaying the functionality of fim regulatory recombinases; based on K137058.

K137058’s invertible repeat sequences responsible for recombinase binding are ordered incorrectly; the regions are not identical, and have “left” and “right” components. K137008 (invertible repeat-right) and K137010 (invertible repeat-left) are mislabeled and belong in the order “K137008-region to invert-K137010” in accordance with the wild-type fim system.

Repeat sequence in this part exist in the correct (wild-type) orientation.

Use of this Part in Characterizing the FimE Recombinase

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

To initially test the FimE recombinase, we utilized a reporter created by the CalTech ‘08 iGEM team (BBa_K137058) for the same purpose. The K137058 reporter in the vector pSB3C5 was co-transformed in 10-beta E. coli (NEB) with strong, constitutively expressed FimE (K880005-K137007) in the vector pSB1A2. Despite several co-transformation trials, no fluorescence was observed in contrast to CalTech ‘08’s results.

Scrutiny of the DNA sequence provided a possible explanation of the negative results. As constructed, verified by sequencing, the orientations of IRL(K137010) and IRR (K137008) within the inversion region of 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 1). We hypothesize that this 180º orientation of the IRL and IRR is the determinative factor to the inversion negative results.

We created a digestible reporter using what we hypothesized are the correct IRL/IRR orientation and got mixed results. See the BBa_K880001 page [1].

To additionally test our hypothesis we made this fluorescent reporter 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[2].

Sequence and Features