Difference between revisions of "Part:BBa K1463000"
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PhiC31 integrase flips the orientation of the DNA between attP and attB, changing the direction of the promoter, switching rfp off and gfp on. | PhiC31 integrase flips the orientation of the DNA between attP and attB, changing the direction of the promoter, switching rfp off and gfp on. | ||
| − | [[Image:switchint.png|thumb|center| | + | [[Image:switchint.png|thumb|center|600px|'''Fig. 1:''' Diagram of the BBa_K1463000 recombination switch. ]] |
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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. | ||
| − | [[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 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.]] | + | [[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.]] |
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. | 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 | + | Sizes of fragments using BamHI are: |
| − | RFP ON GFP OFF (attP and attB) | + | RFP ON GFP OFF (attP and attB) 2339 bp + 2776 bp |
| − | GFP ON RFP OFF (attL and attR) | + | GFP ON RFP OFF (attL and attR) 2477 bp + 2628 bp |
| − | As can be seen, the restriction pattern changes from RFP | + | As can be seen, the restriction pattern changes from RFP ON to GFP ON only when arabinose is added. |
| − | [[Image:Fluoann1.jpg|thumb|center|800px|'''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. ]] | + | [[Image:Fluoann1.jpg|thumb|center|800px|'''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. 200 ul samples of overnight cultures in a 96 well plate scanned using a Typhoon FLA9500 scanner. Overlay of red and green fluorescent images using 532 nm laser and LPG filter and 473 laser and BPB filter respectively. Samples in the middle two rows are duplicate samples of cells corresponding to lanes 3, 4, 6, 7, 9, 10, 2, 5, 8 and 11 in the gel in Fig 2 above. That is the first 6 wells from left to right contain the switch plus pBAD-int treated alternatively with glucose and arabinose, followed by three wells with just switch plasmid on its own (unswitched RFP ON), and a negative control with no RFP or GFP genes.]] |
| − | There is a corresponding switch from red fluorescence to green fluorescence. | + | There is a corresponding switch from red fluorescence to green fluorescence as the orientation of the promoter segment changes under the influence of integrase (Fig. 3). |
Latest revision as of 16:41, 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.
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. 200 ul samples of overnight cultures in a 96 well plate scanned using a Typhoon FLA9500 scanner. Overlay of red and green fluorescent images using 532 nm laser and LPG filter and 473 laser and BPB filter respectively. Samples in the middle two rows are duplicate samples of cells corresponding to lanes 3, 4, 6, 7, 9, 10, 2, 5, 8 and 11 in the gel in Fig 2 above. That is the first 6 wells from left to right contain the switch plus pBAD-int treated alternatively with glucose and arabinose, followed by three wells with just switch plasmid on its own (unswitched RFP ON), and a negative control with no RFP or GFP genes.
There is a corresponding switch from red fluorescence to green fluorescence as the orientation of the promoter segment changes under the influence of integrase (Fig. 3).
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 813
Illegal NheI site found at 836 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 793
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 34
Illegal AgeI site found at 146 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 711
Illegal BsaI.rc site found at 1668