Difference between revisions of "Part:BBa K1463000"

Revision as of 16:23, 17 October 2014

Recombinase Switch With GFP and RFP

A recombinase switch with GFP one side of the switch and RFP on the other.
PhiC31 integrase flips the orientation of the DNA between attP and attB, changing the direction of the promoter, switching rfp off and gfp on.

Fig. 1: Diagram of the BBa_K1463000 recombination switch.

Usage and Biology

Recombination was tested in vivo by placing BBa_K1463000 on a low copy number pSC101 plasmid and introducing a second plasmid (pBAD-int) that only expresses integrase when arabinose is added.

Fig. 2: In vivo recombination of BBa_K1463000 switch using a plasmid (pBAD-Int) that expresses PhiC31 integrase under the control of the arabinose inducible Pbad promoter. Cells were grown for 16 hours with arabinose or glucose. Plasmid DNA was purified, cut with HindIII and run on a 1.2% agarose gel.The gel shows three replicates of the same experiment. 1.pBAD-Int on its own, 2.Switch #2 on its own, 3.Switch #2 + pBAD-Int glucose, 4.Switch #2 + pBAD-Int arabinose, 5.Switch #3 on its own, 6.Switch #3 + pBAD-Int glucose, 7.Switch #3 + pBAD-Int arabinose, 8.Switch #4 on its own, 9.Switch #4 + pBAD-Int glucose, 10.Switch #4 + pBAD-Int arabinose, 11.pBAD33 gvpAC (ignore)12.1kb+ marker.

The HindIII site is placed in such a way that the bands produced by digestion with this restriction enzyme change depending on whether recombination has occurred or not. This pattern can be seen on the gel above.

Sizes of fragments using HindIII and PstI are:

RFP ON GFP OFF (attP and attB) 2339bp + 2776bp

GFP ON RFP OFF (attL and attR) 2628bp + 2477bp

As can be seen, the restriction pattern changes from RFP on to GFP on only when arabinose is added.

Fig. 3: Fluorescence scan showing RFP and GFP fluorescence when BBa_K1463000 recombination switch is grown in vivo with pBAD-int with glucose or arabinose.

There is a corresponding switch from red fluorescence to green fluorescence.

Sequence and Features