Difference between revisions of "Test Mnt/Tet promoter"
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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] | 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] | ||
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| + | pBAD+RBS+TetR+TT=K091145 | ||
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add aTc | add aTc | ||
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I13453+[http://partsregistry.org/Part:BBa_I13025 I13025]+B0015+K091105+E0240 | I13453+[http://partsregistry.org/Part:BBa_I13025 I13025]+B0015+K091105+E0240 | ||
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| + | pBAD+RBS+Mnt+TT=K091147 | ||
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'''Ligation plan for three test sequences''' | '''Ligation plan for three test sequences''' | ||
| − | 1) K091105 ligated with E0240 | + | 1) K091105 (SP) ligated with E0240 (XP) |
| − | 2) | + | 2) ligate pBAD+RBS+TetR+TT (from build XOR gate page) to the 5' end of pMnt/Tet+RBS+GFP |
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| − | |||
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| − | |||
This will make the first promoter test sequence. | This will make the first promoter test sequence. | ||
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| − | 1) ligate the part RBS+Mnt to the 5' end of the TT | + | 1) ligate the part RBS+Mnt (RS) to the 5' end of the TT (RX) |
| − | 2) then ligate pBAD to the 5' end of that | + | 2) then ligate pBAD (RS) to the 5' end of that (RX) |
| − | 3) then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that | + | 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. | ||
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| − | 1) ligate the part RBS+Mnt+TT to the 3' end of pBAD+RBS+TetR | + | 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 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.
