Difference between revisions of "Test pLsrA/cI"

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''' Test pLsrA/cI repression:'''
 
''' Test pLsrA/cI repression:'''
  
−
pBAD→RBS+LsrR+TT
+
pBAD→RBS+lsrK+RBS+LsrR+TT
  
−
plsrA/cI→GFP does not glow
+
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_K091001 K091001]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
+
[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]
 +
 
 +
[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
  
 
add AI-2
 
add AI-2
  
−
pBAD→RBS+LsrR+TT
+
pBAD→RBS+lsrK+RBS+LsrR+TT
  
−
plsrA/cI→GFP glows
+
plsrA/cI→RBS+GFP+TT  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_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091106 K091106]+[http://partsregistry.org/Part:BBa_E0240 E0240]
+
[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]
 +
 
 +
[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 test sequences'''
 
'''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.
  
−
K091106 ligated with E0240
+
Digest pBAD with S/P, RBS+lsrK with X/P; do ligation.
  
−
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)
+
Digest TT with R/X, RBS+lsrR with R/S; do ligation.
  
−
then ligate the promoter+RBS to the 5' end of the lsrR+TT parts together
+
Digest RBS+lsrR+TT with R/X, pBAD+RBS+lsrK with R/S,; do ligation.
  
−
then ligate that part 5' to the plsrA/cI+GFP
+
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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