Difference between revisions of "Test Mnt/Tet promoter"
Line 12: | Line 12: | ||
add aTc | add aTc | ||
− | Some | + | Some promoter→RBS+TetR+t+t+pMnt/Tet→GFP glows |
I13453+RBS+C0040+TT+K091105+E0240 | I13453+RBS+C0040+TT+K091105+E0240 | ||
Line 19: | Line 19: | ||
'''2) Test Mnt repression:''' | '''2) Test Mnt repression:''' | ||
− | Some | + | Some promoter→RBS+Mnt+t+t+pMnt/Tet→GFP glows |
I13453+RBS+C0072+TT+K091105+E0240 | I13453+RBS+C0072+TT+K091105+E0240 | ||
Line 26: | Line 26: | ||
'''3) Test Tet and Mnt repression:''' | '''3) Test Tet and Mnt repression:''' | ||
− | Some | + | Some promoter→RBS+TetR+Mnt+t+t+pMnt/Tet→GFP does not glows |
I13453+RBS+C0040+C0072+TT+K091105+E0240 | I13453+RBS+C0040+C0072+TT+K091105+E0240 | ||
Line 33: | Line 33: | ||
− | ''' | + | '''Ligation plan for three test sequences''' |
K091105 ligated with E0240 | K091105 ligated with E0240 | ||
Line 39: | Line 39: | ||
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) | 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 | + | then ligate the promoter+RBS to the 5' end of the TetR+TT parts together |
− | then ligate that part | + | then ligate that part 5' to the pMnt/Tet+GFP |
+ | |||
+ | This will make the first promoter test sequence. | ||
+ | |||
+ | |||
+ | |||
+ | ligate Mnt to the double terminator | ||
+ | |||
+ | the ligate the promoter+RBS from the first test sequence to the 5' end of the Mnt+TT | ||
+ | |||
+ | ligate this to the 5' end of the pMnt/Tet+GFP | ||
+ | |||
+ | This makes the second promoter test sequence. | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | ligate the Mnt+TT from the second test sequence to the 3' end of the TetR coding region | ||
+ | |||
+ | 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 | ||
+ | |||
+ | |||
+ | 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. |
Revision as of 19:54, 30 June 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 repress the promoter causing it not glow.
1) Test tet repression:
Some promoter (pbad)→RBS+TetR+t+t+pMnt/Tet→RBS+GFP does not glows
I13453+RBS+C0040+TT+K091105+E0240
add aTc
Some promoter→RBS+TetR+t+t+pMnt/Tet→GFP glows
I13453+RBS+C0040+TT+K091105+E0240
2) Test Mnt repression:
Some promoter→RBS+Mnt+t+t+pMnt/Tet→GFP glows
I13453+RBS+C0072+TT+K091105+E0240
3) Test Tet and Mnt repression:
Some promoter→RBS+TetR+Mnt+t+t+pMnt/Tet→GFP does not glows
I13453+RBS+C0040+C0072+TT+K091105+E0240
add aTc does not glow
Ligation plan for three test sequences
K091105 ligated with E0240
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
This will make the first promoter test sequence.
ligate Mnt to the double terminator
the ligate the promoter+RBS from the first test sequence to the 5' end of the Mnt+TT
ligate this to the 5' end of the pMnt/Tet+GFP
This makes the second promoter test sequence.
ligate the Mnt+TT from the second test sequence to the 3' end of the TetR coding region
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
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.