Test pLasR/cI
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.
| LasR | cI | p-AI-1 + GFP | p-AI-1 - GFP |
|---|---|---|---|
| 0 | 0 | 0 | 0 |
| 1 | 0 | 1 | 0 |
| 0 | 1 | 0 | 0 |
| 1 | 1 | 0 | 0 |
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 glows
I13453+B0034+C0051+B0015+K09110?+E0240
add cI protein
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.
