Difference between revisions of "Test Mnt/Tet promoter"

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.