Difference between revisions of "Test Mnt/Tet promoter"
Line 12: | Line 12: | ||
add aTc | add aTc | ||
− | pBAD→RBS+TetR+TT+pMnt/ | + | pBAD→RBS+TetR+TT+pMnt/Tet→RBS+GFP glows |
I13453+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_C0040 C0040]+B0015+K091105+E0240 | I13453+[http://partsregistry.org/Part:BBa_B0034 B0034]+[http://partsregistry.org/Part:BBa_C0040 C0040]+B0015+K091105+E0240 | ||
Line 19: | Line 19: | ||
'''2) Test Mnt repression:''' | '''2) Test Mnt repression:''' | ||
− | pBAD→RBS+Mnt+TT+pMnt/ | + | pBAD→RBS+Mnt+TT+pMnt/Tet→RBS+GFP glows |
− | I13453 | + | I13453+[http://partsregistry.org/Part:BBa_I13025 I13025]+B0015+K091105+E0240 |
'''3) Test Tet and Mnt repression:''' | '''3) Test Tet and Mnt repression:''' | ||
− | pBAD→RBS+TetR+RBS+Mnt+TT+pMnt/ | + | pBAD→RBS+TetR+RBS+Mnt+TT+pMnt/Tet→RBS+GFP does not glows |
I13453+B0034+C0040+B0034+C0072+B0015+K091105+E0240 | I13453+B0034+C0040+B0034+C0072+B0015+K091105+E0240 | ||
Line 37: | Line 37: | ||
K091105 ligated with E0240 | K091105 ligated with E0240 | ||
− | need to ligate | + | need to ligate pBAD to and RBS |
− | then ligate the | + | then ligate TetR to the 3' end of that |
+ | |||
+ | then ligate TT to the 3' end of that | ||
then ligate that part 5' to the pMnt/Tet+GFP | then ligate that part 5' to the pMnt/Tet+GFP | ||
Line 47: | Line 49: | ||
− | ligate Mnt to the | + | ligate the part RBS+Mnt to the 5' end of the TT |
− | + | then ligate pBAD to the 5' end of that | |
− | ligate | + | 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 57: | Line 59: | ||
+ | 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 | |
− | |||
− | |||
− | then ligate the | ||
− | |||
− | |||
This makes the third promoter test sequence | This makes the third promoter test sequence |
Revision as of 19:49, 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
K091105 ligated with E0240
need to ligate pBAD to and RBS
then ligate TetR to the 3' end of that
then ligate TT to the 3' end of that
then ligate that part 5' to the pMnt/Tet+GFP
This will make the first promoter test sequence.
ligate the part RBS+Mnt to the 5' end of the TT
then ligate pBAD to the 5' end of that
then ligate the part pMnt/Tet+RBS+GFP to the 3' end of that
This makes the second promoter test sequence.
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
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.