Difference between revisions of "Test pLsrA/cI"

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'''1)Control group'''
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''' Test pLsrA/cI repression:'''
  
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plsrA/cI→GFP  glows
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pBAD→RBS+lsrK+RBS+LsrR+TT
  
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[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
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plsrA/cI→RBS+GFP+TT does not glow
  
 +
[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091002 K091002]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091001 K091001]+[http://partsregistry.org/Part:BBa_B0015 B0015]
  
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'''2) Test LsrR repression:'''
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[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
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pBAD→RBS+LsrR+TT+plsrA/cI→GFP does not glow
 
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[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091001 K091001]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
 
  
 
add AI-2
 
add AI-2
  
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pBAD→RBS+LsrR+TT+plsrA/cI→GFP glows
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pBAD→RBS+lsrK+RBS+LsrR+TT
  
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[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091001 K091001]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
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plsrA/cI→RBS+GFP+TT  glows
  
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'''3) Test cI repression:'''
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[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091002 K091002]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091001 K091001]+[http://partsregistry.org/Part:BBa_B0015 B0015]
  
−
pBAD→RBS+cI+TT+plsrA/cI→GFP does not glow
+
[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
−
 
 
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[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_C0051 C0051]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
 
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'''4) Test LsrR and cI repression:'''
 
−
 
 
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pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows
 
−
 
 
−
[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091001 K091001]+[http://partsregistry.org/Part:BBa_C0051 C0051]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
 
−
 
 
−
add AI-2
 
−
 
 
−
pBAD→RBS+lsrR+cI+TT+plsrA/cI→GFP does not glows
 
−
 
 
−
[http://partsregistry.org/Part:BBa_I13453 I13453]+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_K091001 K091001]+[http://partsregistry.org/Part:BBa_C0051 C0051]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
 
  
 
{| border="1" cellpadding="2"
 
{| border="1" cellpadding="2"
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'''Ligation plan for three test sequences'''
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'''Ligation plan for test sequences'''
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1)
 
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K091106 ligated with E0240
 
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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)
 
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2)
 
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K091106 ligated with E0240
 
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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)
 
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then ligate the promoter+RBS to the 5' end of the lsrR+TT parts together
 
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then ligate that part 5' to the plsrA/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 plsrA/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 lsrR 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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Digest RBS with S/P, lsrK with X/P; do ligation.
  
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next ligate the plsrA/cI to the 3' end of that entire sequence
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Digest RBS with S/P, lsrR with X/P; do ligation.
  
 +
Digest pBAD with S/P, RBS+lsrK with X/P; do ligation.
  
 +
Digest TT with R/X, RBS+lsrR with R/S; do ligation.
  
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the 2) and 4) test sequences will also need to be tested by adding AI-2
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Digest RBS+lsrR+TT with R/X, pBAD+RBS+lsrK with R/S,; do ligation.
  
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the 2) test sequence should glow and the 4) should not.
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Digest plsrA/cI with S/P, E0240 with X/P; do ligation.

Latest revision as of 14:54, 11 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.


Test pLsrA/cI repression:

pBAD→RBS+lsrK+RBS+LsrR+TT

plsrA/cI→RBS+GFP+TT does not glow

I13453+B0034+K091002+B0034+K091001+B0015

K091106+E0240

add AI-2

pBAD→RBS+lsrK+RBS+LsrR+TT

plsrA/cI→RBS+GFP+TT glows

I13453+B0034+K091002+B0034+K091001+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 test sequences

Digest RBS with S/P, lsrK with X/P; do ligation.

Digest RBS with S/P, lsrR with X/P; do ligation.

Digest pBAD with S/P, RBS+lsrK with X/P; do ligation.

Digest TT with R/X, RBS+lsrR with R/S; do ligation.

Digest RBS+lsrR+TT with R/X, pBAD+RBS+lsrK with R/S,; do ligation.

Digest plsrA/cI with S/P, E0240 with X/P; do ligation.

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