Difference between revisions of "Part:BBa K1463000"
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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.  
 
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.  
  
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[[Image:Switchgelann.jpg|thumb|center|500px|'''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.]]
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[[Image:Switchgelann.jpg|thumb|center|500px|'''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 BamHI, which cuts once in the switch and once in the vector, 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.]]
  
  
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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.  
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The BamHI sites are 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.  
  
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Sizes of fragments using HindIII and PstI are:
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Sizes of fragments using BamHI are:
  
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RFP ON GFP OFF (attP and attB) 2339bp + 2776bp
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RFP ON GFP OFF (attP and attB) 2339 bp + 2776 bp
  
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GFP ON RFP OFF (attL and attR) 2628bp + 2477bp
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GFP ON RFP OFF (attL and attR) 2477 bp + 2628 bp
  
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As can be seen, the restriction pattern changes from RFP on to GFP on only when arabinose is added.
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As can be seen, the restriction pattern changes from RFP ON to GFP ON only when arabinose is added.
  
  

Revision as of 16:28, 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 BamHI, which cuts once in the switch and once in the vector, 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 BamHI sites are 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 BamHI are:

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

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

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 813
    Illegal NheI site found at 836
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 793
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 34
    Illegal AgeI site found at 146
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 711
    Illegal BsaI.rc site found at 1668