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| − | 1)Control group
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| | | | |
| − | plasR/cI→RBS+GFP does not glow
| + | '''Test pLasR/cI:''' |
| | | | |
| − | K09110?+E0240
| + | pBAD→RBS+LasR+TT (from James DC) |
| | | | |
| | + | I13453+B0034+C0079+B0015 |
| | | | |
| − | 2) Test LasR activation:
| + | plasR/cI→RBS+GFP+tt |
| | | | |
| − | pBAD→RBS+LasR+t+t+plsrA/cI→RBS+GFP does not glow
| + | K09110?+E0240 does not glow |
| | | | |
| − | I13453+B0034+C0079+B0015+K09110?+E0240
| + | '''add P-AI-1''' |
| | | | |
| − | add P-AI-1
| + | pBAD→RBS+LasR+TT (from James DC) |
| | | | |
| − | pBAD→RBS+LsrR+t+t+plsrA/cI→RBS+GFP glows
| + | I13453+B0034+C0079+B0015 |
| | | | |
| − | I13453+B0034+C0079+B0015+K091106+E0240
| + | plasR/cI→RBS+GFP+tt |
| | | | |
| | + | K09110?+E0240 glows |
| | | | |
| − | 3) Test cI repression:
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| | | | |
| − | pBAD→RBS+cI+TT+plasR/cI→GFP glows
| + | '''Ligation plan for test sequences''' |
| | | | |
| − | I13453+B0034+C0051+B0015+K09110?+E0240
| + | Digest plasR/cI with S/P, E0240 with X/P; do ligation. |
| − | | |
| − | add cI protein
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| − | | |
| − | pBAD→RBS+cI+TT+plasR/cI→GFP does not glow
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| − | | |
| − | I13453+B0034+C0051+B0015+K09110?+E0240
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| − | | |
| − | | |
| − | 4) Test lasR activation VS cI repression :
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| − | | |
| − | pBAD→RBS+lasR+cI+TT+plsrA/cI→GFP does not glows
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| − | | |
| − | I13453+B0034+C0079+C0051+B0015+K09110?+E0240
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| − | | |
| − | add P-AI-1
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| − | | |
| − | pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows
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| − | | |
| − | I13453+B0034+C0079+C0051+B0015+K09110?+E0240
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| − | | |
| − | | |
| − | | |
| − | Ligation plan for three test sequences
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| − | | |
| − | 1)
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| − | | |
| − | K09110? ligated with E0240
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| − | | |
| − | 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)
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| − | | |
| − | | |
| − | 2)
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| − | | |
| − | K09110? ligated with E0240
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| − | | |
| − | 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)
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| − | | |
| − | then ligate the promoter+RBS to the 5' end of the lasR+TT parts together
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| − | | |
| − | then ligate that part 5' to the plasR/cI+GFP
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| − | | |
| − | | |
| − | 3)
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| − | | |
| − | ligate cI to the double terminator
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| − | | |
| − | the ligate the promoter+RBS from the first test sequence to the 5' end of the cI+TT
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| − | | |
| − | ligate this to the 5' end of the plasR/cI+GFP
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| − | | |
| − | | |
| − | 4)
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| − | | |
| − | ligate the cI+TT from the second test sequence to the 3' end of the lasR coding region
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| − | | |
| − | then ligate the promoter+RBS from the first test sequence to the 5' end of that
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| − | | |
| − | next ligate the plasA/cI to the 3' end of that entire sequence
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| − | | |
| − | | |
| − | the 2) and 4) test sequences will also need to be tested by adding P-AI-1
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| − | | |
| − | the 2) test sequence should glow and the 4) should not.
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Latest revision as of 14:54, 11 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.
| 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
|
Test pLasR/cI:
pBAD→RBS+LasR+TT (from James DC)
I13453+B0034+C0079+B0015
plasR/cI→RBS+GFP+tt
K09110?+E0240 does not glow
add P-AI-1
pBAD→RBS+LasR+TT (from James DC)
I13453+B0034+C0079+B0015
plasR/cI→RBS+GFP+tt
K09110?+E0240 glows
Ligation plan for test sequences
Digest plasR/cI with S/P, E0240 with X/P; do ligation.
