Test Mnt/Tet promoter

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.

TetR	Mnt	aTc + GFP	aTc - GFP
0	0	1	1
0	1	0	0
1	0	1	0
1	1	0	0

1) Test tet repression:

pBAD→RBS+TetR+t+t+pMnt/Tet→RBS+GFP does not glows

I13453+S0101+B0015+K091105+E0240

pBAD+RBS+TetR+TT=K091145

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

pBAD+RBS+Mnt+TT=K091147

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 (SP) ligated with E0240 (XP)

2) ligate pBAD+RBS+TetR+TT (from build XOR gate page) to the 5' end of pMnt/Tet+RBS+GFP

This will make the first promoter test sequence.

1) ligate the part RBS+Mnt (RS) to the 5' end of the TT (RX)

2) then ligate pBAD (RS) to the 5' end of that (RX)

3) then ligate the part pMnt/Tet+RBS+GFP (RS) to the 3' end of that (RX)

This makes the second promoter test sequence.

1) ligate the part RBS+Mnt+TT (RX) to the 3' end of pBAD+RBS+TetR (RS)

2) then ligate the part pMnt/Tet+RBS+GFP (SP) to the 3' end of that (XP)

This makes the third promoter test sequence

These test sequences should be inserted into pSB4A3

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.