Difference between revisions of "Test Mnt/Tet promoter"
| Line 35: | Line 35: | ||
'''Ligation plan for three test sequences''' | '''Ligation plan for three test sequences''' | ||
| − | K091105 ligated with E0240 | + | 1) K091105 ligated with E0240 |
| − | need to ligate pBAD to and RBS | + | 2) need to ligate pBAD to and RBS |
| − | then ligate TetR to the 3' end of that | + | 3) then ligate TetR to the 3' end of that |
| − | then ligate TT to the 3' end of that | + | 4)then ligate TT to the 3' end of that |
| − | then ligate that part 5' to the pMnt/Tet+GFP | + | 5)then ligate that part 5' to the pMnt/Tet+GFP |
This will make the first promoter test sequence. | This will make the first promoter test sequence. | ||
| Line 49: | Line 49: | ||
| − | ligate the part RBS+Mnt to the 5' end of the TT | + | 1) ligate the part RBS+Mnt to the 5' end of the TT |
| − | then ligate pBAD to the 5' end of that | + | 2) then ligate pBAD to the 5' end of that |
| − | then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that | + | 3) then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that |
This makes the second promoter test sequence. | This makes the second promoter test sequence. | ||
| Line 59: | Line 59: | ||
| − | ligate the part RBS+Mnt+TT to the 3' end of pBAD+RBS+TetR | + | 1) ligate the part RBS+Mnt+TT to the 3' end of pBAD+RBS+TetR |
| − | then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that | + | 2) then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that |
| − | This makes the third promoter test sequence | + | 3) This makes the third promoter test sequence |
Revision as of 19:53, 2 July 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.
1) Test tet repression:
pBAD→RBS+TetR+t+t+pMnt/Tet→RBS+GFP does not glows
I13453+S0101+B0015+K091105+E0240
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
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 ligated with E0240
2) need to ligate pBAD to and RBS
3) then ligate TetR to the 3' end of that
4)then ligate TT to the 3' end of that
5)then ligate that part 5' to the pMnt/Tet+GFP
This will make the first promoter test sequence.
1) ligate the part RBS+Mnt to the 5' end of the TT
2) then ligate pBAD to the 5' end of that
3) then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that
This makes the second promoter test sequence.
1) ligate the part RBS+Mnt+TT to the 3' end of pBAD+RBS+TetR
2) then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that
3) This makes the third promoter test sequence
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.
