Difference between revisions of "Test pLasR/cI"

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1)Control group
 
  
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:
 
  
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
 
 
 
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.
 

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.