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.
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→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+RBS+Mnt+TT+pMnt/Tet→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 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.
Parts Needed for XOR Gate:
| part no. | description | source | responsible |
|---|---|---|---|
| K091105 | pMnt/Tet | MW '08 | RC |
| B0034 | RBS | registry | ? |
| C0061 | Lux gene(+LVA) | registry | ? |
| C0062 | Lux gene(-LVA) | registry | ? |
| C0051 | cI gene | registry | ? |
| B0015 | TT | registry | ? |
Steps Needed for Test XOR Gate:
| step no. | vector BBa | vector description | vector suffix prefix | vector enzymes | vector size (bp) | insert BBa | insert description | insert suffix prefix | insert enzymes | insert size (bp) | product BBa | product size |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | B0034 | RBS | suffix | SP | 2100 | C0061 | Lux (+LVA) | suffix, prefix | XP | 756 | ? | 2850 |
| 2 | B0034 | RBS | suffix | SP | 2100 | C0051 | cI | suffix, prefix | XP | 750 | ? | 2850 |
| 3 | product from step 1 | RBS+ Lux | suffix | SP | 2850 | product from step 2 | RBS+ cI | suffix, prefix | XP | 762 | ? | 3612 |
| 4 | product from step 3 | RBS+Lux+ RBS+cI | suffix, prefix | RS | 3612 | B0015 | TT | prefix | RX | 2221 | ? | 3751 |
| 5 | product from step 4 | RBS+Lux+RBS+cI+TT | suffix prefix | XP | 3751 | K091105 | pMnt/Tet | suffix | SP | 2200 | ? | 3949 |
