Difference between revisions of "Test Mnt/Tet promoter"
Line 8: | Line 8: | ||
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_B0034 B0034]+[http://partsregistry.org/Part:BBa_C0040 C0040]+[http://partsregistry.org/Part:BBa_B0015 B0015]+[http://partsregistry.org/Part:BBa_K091105 K091105]+[http://partsregistry.org/Part:BBa_E0240 E0240] |
add aTc | add aTc | ||
Line 21: | Line 21: | ||
pBAD→RBS+Mnt+TT+pMnt/Tet→GFP glows | pBAD→RBS+Mnt+TT+pMnt/Tet→GFP glows | ||
− | I13453+B0034+C0072+B0015+K091105+E0240 | + | I13453+B0034+[http://partsregistry.org/Part:BBa_C0072 C0072]+B0015+K091105+E0240 |
Revision as of 14:38, 1 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+B0034+C0040+B0015+K091105+E0240
add aTc
pBAD→RBS+TetR+TT+pMnt/Tet→GFP glows
I13453+B0034+C0040+B0015+K091105+E0240
2) Test Mnt repression:
pBAD→RBS+Mnt+TT+pMnt/Tet→GFP glows
I13453+B0034+C0072+B0015+K091105+E0240
3) Test Tet and Mnt repression:
pBAD→RBS+TetR+Mnt+TT+pMnt/Tet→GFP does not glows
I13453+B0034+C0040+C0072+B0015+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.