Difference between revisions of "Test Mnt/Tet promoter"

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Test Mnt/Tet promoter
 
Test Mnt/Tet promoter
  
The pMnt/TetR promoter is constitutively on and is repressed by the TetR protein. The addition of aTc should repress the TetR causing it to glow. The addition of the Mnt protein should repress the promoter causing it not glow.
+
The pMnt/TetR promoter is constitutively on and is repressed by the TetR protein. The addition of aTc should repress the TetR causing it to glow. The addition of the Mnt protein should also repress the promoter, causing it to not glow.
 +
 
 +
{| border="1" cellpadding="2"
 +
!width="30"|TetR
 +
!width="30"|Mnt
 +
!width="30"|aTc + GFP
 +
!width="30"|aTc - GFP
 +
|-
 +
| 0 || 0 || 1 || 1
 +
|-
 +
| 0 || 1 || 0 || 0
 +
|-
 +
| 1 || 0 || 1 || 0
 +
|-
 +
| 1 || 1 || 0 || 0
 +
|}
 +
 
  
  
Line 8: Line 24:
 
pBAD→RBS+TetR+t+t+pMnt/Tet→RBS+GFP does not glows
 
pBAD→RBS+TetR+t+t+pMnt/Tet→RBS+GFP does not glows
  
I13453+B0034+C0040+B0015+K091105+E0240
+
I13453+[http://partsregistry.org/Part:BBa_S0101 S0101]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091105 K091105]+[http://partsregistry.org/Part:BBa_E0240 E0240]
 +
 
 +
 
 +
pBAD+RBS+TetR+TT=K091145
 +
 
  
 
add aTc
 
add aTc
  
pBAD→RBS+TetR+TT+pMnt/Tet→GFP glows
+
pBAD→RBS+TetR+TT+pMnt/Tet→RBS+GFP glows
  
I13453+B0034+C0040+B0015+K091105+E0240
+
I13453+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_C0040 C0040]+B0015+K091105+E0240
  
  
 
'''2) Test Mnt repression:'''
 
'''2) Test Mnt repression:'''
  
pBAD→RBS+Mnt+TT+pMnt/Tet→GFP glows
+
pBAD→RBS+Mnt+TT+pMnt/Tet→RBS+GFP glows
 +
 
 +
I13453+[http://partsregistry.org/Part:BBa_I13025 I13025]+B0015+K091105+E0240
 +
 
 +
 
 +
pBAD+RBS+Mnt+TT=K091147
  
I13453+B0034+C0072+B0015+K091105+E0240
 
  
  
 
'''3) Test Tet and Mnt repression:'''
 
'''3) Test Tet and Mnt repression:'''
  
pBAD→RBS+TetR+Mnt+TT+pMnt/Tet→GFP does not glows
+
pBAD→RBS+TetR+RBS+Mnt+TT+pMnt/Tet→RBS+GFP does not glows
  
I13453+B0034+C0040+C0072+B0015+K091105+E0240
+
I13453+B0034+C0040+B0034+C0072+B0015+K091105+E0240
  
 
add aTc does not glow
 
add aTc does not glow
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'''Ligation plan for three test sequences'''
 
'''Ligation plan for three test sequences'''
  
K091105 ligated with E0240
+
1) K091105 (SP) ligated with E0240 (XP)
 
 
need to ligate some promoter (digest with E and S) with an RBS (digest E and X) and ligate TetR(digest with E and S) with a double terminator (digest with E and x)
 
 
 
then ligate the promoter+RBS to the 5' end of the TetR+TT parts together
 
  
then ligate that part 5' to the pMnt/Tet+GFP
+
2) ligate pBAD+RBS+TetR+TT (from build XOR gate page) to the 5' end of pMnt/Tet+RBS+GFP
  
 
This will make the first promoter test sequence.
 
This will make the first promoter test sequence.
Line 47: Line 67:
  
  
ligate Mnt to the double terminator
+
1) ligate the part RBS+Mnt (RS) to the 5' end of the TT (RX)
  
the ligate the promoter+RBS from the first test sequence to the 5' end of the Mnt+TT
+
2) then ligate pBAD (RS) to the 5' end of that (RX)
  
ligate this to the 5' end of the pMnt/Tet+GFP
+
3) then ligate the part pMnt/Tet+RBS+GFP (RS) to the 3' end of that (RX)
  
 
This makes the second promoter test sequence.
 
This makes the second promoter test sequence.
Line 57: Line 77:
  
  
 +
1) ligate the part RBS+Mnt+TT (RX) to the 3' end of pBAD+RBS+TetR (RS)
  
 +
2) then ligate the part pMnt/Tet+RBS+GFP (SP) to the 3' end of that (XP)
  
ligate the Mnt+TT from the second test sequence to the 3' end of the TetR coding region
+
This makes the third promoter test sequence
  
then ligate the promoter+RBS from the first test sequence to the 5' end of that
 
 
next ligate the pMnt/Tet to the 3' end of that entire sequence
 
 
This makes the third promoter test sequence
 
  
 +
These test sequences should be inserted into pSB4A3
  
 
the first and third test sequences will also need to be tested by adding aTc  
 
the first and third test sequences will also need to be tested by adding aTc  
  
 
the first test sequence should glow and the third should not.
 
the first test sequence should glow and the third should not.

Latest revision as of 16:43, 4 August 2008

Test Mnt/Tet promoter

The pMnt/TetR promoter is constitutively on and is repressed by the TetR protein. The addition of aTc should repress the TetR causing it to glow. The addition of the Mnt protein should also repress the promoter, causing it to not glow.

TetR Mnt aTc + GFP aTc - GFP
0 0 1 1
0 1 0 0
1 0 1 0
1 1 0 0


1) Test tet repression:

pBAD→RBS+TetR+t+t+pMnt/Tet→RBS+GFP does not glows

I13453+S0101+B0015+K091105+E0240


pBAD+RBS+TetR+TT=K091145


add aTc

pBAD→RBS+TetR+TT+pMnt/Tet→RBS+GFP glows

I13453+B0034+C0040+B0015+K091105+E0240


2) Test Mnt repression:

pBAD→RBS+Mnt+TT+pMnt/Tet→RBS+GFP glows

I13453+I13025+B0015+K091105+E0240


pBAD+RBS+Mnt+TT=K091147


3) Test Tet and Mnt repression:

pBAD→RBS+TetR+RBS+Mnt+TT+pMnt/Tet→RBS+GFP does not glows

I13453+B0034+C0040+B0034+C0072+B0015+K091105+E0240

add aTc does not glow


Ligation plan for three test sequences

1) K091105 (SP) ligated with E0240 (XP)

2) ligate pBAD+RBS+TetR+TT (from build XOR gate page) to the 5' end of pMnt/Tet+RBS+GFP

This will make the first promoter test sequence.


1) ligate the part RBS+Mnt (RS) to the 5' end of the TT (RX)

2) then ligate pBAD (RS) to the 5' end of that (RX)

3) then ligate the part pMnt/Tet+RBS+GFP (RS) to the 3' end of that (RX)

This makes the second promoter test sequence.


1) ligate the part RBS+Mnt+TT (RX) to the 3' end of pBAD+RBS+TetR (RS)

2) then ligate the part pMnt/Tet+RBS+GFP (SP) to the 3' end of that (XP)

This makes the third promoter test sequence


These test sequences should be inserted into pSB4A3

the first and third test sequences will also need to be tested by adding aTc

the first test sequence should glow and the third should not.