Difference between revisions of "Part:BBa K1463000"

Revision as of 12:44, 15 October 2014

Recombinase Switch With GFP and RFP

A recombinase switch with GFP one side of the switch and RFP on the other.

Fig. 1: 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.

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 BamHI fragments are:

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

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

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

@@ Line 4: / Line 4: @@
 A recombinase switch with GFP one side of the switch and RFP on the other.
-[[Image:GU_jake_gel_1.jpg|thumb|500px|left|'''Fig. 1:''' 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.]]
+https://static.igem.org/mediawiki/2014/f/f6/Switch_diagram_sites_small2.jpg
+[[Image:GU_jake_gel_1.jpg|thumb|500px|right|'''Fig. 1:''' 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.]]
-https://static.igem.org/mediawiki/2014/f/f3/GU_jake_gel_1.jpg
-The image below shows a schematic of the switch. 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 BamHI fragments are:
@@ Line 20: / Line 20: @@
-https://static.igem.org/mediawiki/2014/f/f6/Switch_diagram_sites_small2.jpg
 <!-- Add more about the biology of this part here