Difference between revisions of "Part:BBa K1463002"

Revision as of 19:59, 17 October 2014

Recombinase Switch With GFP (weaker RBS)

PhiC31-based recombinase switch with GFP to the right and a leftward facing promoter. PhiC31 integrase switches on GFP expression. GFP contains a weaker ribosome binding site than in BBa_K1463001.

Recombination was tested in vitro by combining plasmid DNA containing the part with purified phiC31 integrase, which causes the DNA sequence to invert. The HindIII sites are placed in such a way that the bands produced by restriction digest, together with PstI, change depending on whether inversion has occured or not. This pattern can be seen in the gel (figure 1).

After the recombination reaction the promoter is orientated in the correct direction to drive expression of GFP. This can be seen when the plasmid is transformed into cells. Figure 2 shows the difference between colonies expressing GFP and those which are not. Also shown in figure 2 is the comparison with part BBa_K1463001, where it can be seen the weaker ribosome binding site produces less brightly fluorescing colonies.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 82
Illegal NheI site found at 105
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 62
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 938

@@ Line 3: / Line 3: @@
 PhiC31-based recombinase switch with GFP to the right and a leftward facing promoter. PhiC31 integrase switches on GFP expression. GFP contains a weaker ribosome binding site than in [https://parts.igem.org/Part:BBa_K1463001 BBa_K1463001].
+Recombination was tested in vitro by combining plasmid DNA containing the part with purified phiC31 integrase, which causes the DNA sequence to invert. The HindIII sites are placed in such a way that the bands produced by restriction digest, together with PstI, change depending on whether inversion has occured or not. This pattern can be seen in the gel (figure 1).
+After the recombination reaction the promoter is orientated in the correct direction to drive expression of GFP. This can be seen when the plasmid is transformed into cells. Figure 2 shows the difference between colonies expressing GFP and those which are not. Also shown in figure 2 is the comparison with part [https://parts.igem.org/Part:BBa_K1463001 BBa_K1463001], where it can be seen the weaker ribosome binding site produces less brightly fluorescing colonies.
 <!-- Add more about the biology of this part here