Difference between revisions of "Test pLasR/cI"

Revision as of 14:53, 1 July 2008

pLasR/cI is constitutively off and is repressed by cI. The addition of p-AI-1, activates LasR causing the promoter to turn on. The addition of cI to should repress the promoter causing it to turn off.

1)Control group

plasR/cI→RBS+GFP does not glow

K09110?+E0240

2) Test LasR activation:

pBAD→RBS+LasR+t+t+plsrA/cI→RBS+GFP does not glow

I13453+B0034+C0079+B0015+K09110?+E0240

add P-AI-1

pBAD→RBS+LsrR+t+t+plsrA/cI→RBS+GFP glows

I13453+B0034+C0079+B0015+K091106+E0240

3) Test cI repression:

pBAD→RBS+cI+TT+plasR/cI→GFP does not glow

I13453+B0034+C0051+B0015+K09110?+E0240

4) Test lasR activation VS cI repression :

pBAD→RBS+lasR+cI+TT+plsrA/cI→GFP does not glows

I13453+B0034+C0079+C0051+B0015+K09110?+E0240

add P-AI-1

pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows

I13453+B0034+C0079+C0051+B0015+K09110?+E0240

Ligation plan for three test sequences

1)

K09110? ligated with E0240

need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lasR(digest with E/S) with a double terminator (digest with E/X)

2)

K09110? ligated with E0240

need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lasR(digest with E/S) with a double terminator (digest with E/X)

then ligate the promoter+RBS to the 5' end of the lasR+TT parts together

then ligate that part 5' to the plasR/cI+GFP

3)

ligate cI to the double terminator

the ligate the promoter+RBS from the first test sequence to the 5' end of the cI+TT

ligate this to the 5' end of the plasR/cI+GFP

4)

ligate the cI+TT from the second test sequence to the 3' end of the lasR coding region

then ligate the promoter+RBS from the first test sequence to the 5' end of that

next ligate the plasA/cI to the 3' end of that entire sequence

the 2) and 4) test sequences will also need to be tested by adding P-AI-1

the 2) test sequence should glow and the 4) should not.