Difference between revisions of "Test pLsrA/cI"
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Revision as of 14:40, 1 July 2008
Test pLsrA/cI promoter
The pLsrA/cI promoter is constitutively off and is repressed by the lsrR protein. The addition of AI-2 should de-repress the lsrR operon causing it to glow. The addition of the cI protein should repress the promoter, causing it to not glow.
1)Control group
plsrA/cI→RBS+GFP glows
K091106+E0240
2) Test LsrR repression:
pBAD→RBS+LsrR+t+t+plsrA/cI→RBS+GFP does not glow
I13453+B0034+K091001+B0015+K091106+E0240
add AI-2
pBAD→RBS+LsrR+t+t+plsrA/cI→RBS+GFP glows
I13453+B0034+K091001+B0015+K091106+E0240
3) Test cI repression:
pBAD→RBS+cI+TT+plsrA/cI→GFP glows
I13453+B0034+C0051+B0015+K091106+E0240
add cI protein
pBAD→RBS+cI+TT+plsrA/cI→GFP does not glow
I13453+B0034+C0051+B0015+K091106+E0240
4) Test LsrR and cI repression:
pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows
I13453+B0034+K091001+C0051+B0015+K091106+E0240
add AI-2
pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows
I13453+B0034+K091001+C0051+B0015+K091106+E0240
| LsrR | cI | AI-2 + GFP | AI-2 - GFP |
|---|---|---|---|
| 0 | 0 | 1 | 1 |
| 1 | 0 | 1 | 0 |
| 0 | 1 | 0 | 0 |
| 1 | 1 | 0 | 0 |
Ligation plan for three test sequences
1)
K091106 ligated with E0240
need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lsrR(digest with E/S) with a double terminator (digest with E/X)
2)
K091106 ligated with E0240
need to ligate some promoter (digest with E/S) with an RBS (digest E/X) and ligate lsrR(digest with E/S) with a double terminator (digest with E/X)
then ligate the promoter+RBS to the 5' end of the lsrR+TT parts together
then ligate that part 5' to the plsrA/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 plsrA/cI+GFP
4)
ligate the cI+TT from the second test sequence to the 3' end of the lsrR coding region
then ligate the promoter+RBS from the first test sequence to the 5' end of that
next ligate the plsrA/cI to the 3' end of that entire sequence
the 2) and 4) test sequences will also need to be tested by adding AI-2
the 2) test sequence should glow and the 4) should not.
