Difference between revisions of "Test pLasR/cI"

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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.

Revision as of 14:51, 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.

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.

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