Test XOR Hybrid Promoters

This is a rough idea of how to test the four hybrid promoters for the XOR gate.

Test pLuxR/cI

luxR/CI hybrid promoter is constitutively off. Ligate with GFP on the 3’ end. If luxR is present first then add AI-1, the bacteria should glow green. Then add CI, the bacteria should not glow.

Some promoter→LuxR+t+t+pLuxR/cI→GFP does not glows

add AI-1

Some promoter→LuxR+t+t+pLuxR/cI→GFP glows

add AI-1

Some promoter→LuxR+cI+t+t+pLuxR/cI→GFP does not glow

Test pMnt/Lac pMnt/Lac is constitutively on but is repressed by the LacI protein. The promoter is also repressed by the Mnt protein. The addition of IPTG should repress the LacI repressor and glow in the absence of the Mnt protein. Some promoter→LacI+t+t+pLac/Mnt→GFP does not glow Some promoter→Mnt+t+t+pLac/Mnt→GFP does not glow add IPTG Some promoter→LacI+t+t+pLac/Mnt→GFP glows Test pLsrR/cI pLsrR/cI is constitutively on and is repressed by LsrR. The addition of AI-2, which becomes phosphorylated by LsrK, represses LsrR causing the promoter to turn on. The addition of cI to should repress the promoter causing it to turn off. Some promoter→LsrK+LsrR+t+t+pLsrR/cI→GFP does not glow add AI-2 Some promoter→LsrK+LsrR+t+t+pLsrR/cI→GFP glows add AI-2 Some promoter→LsrK+LsrR+cI+t+t+pLsrR/cI→GFP does not glows Test pMnt/Tet 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. 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→TetR+t+t+pMnt/Tet→GFP glows I13453+RBS+C0040+TT+K091105+E0240 Test Mnt repression: Some promoter→Mnt+t+t+pMnt/Tet→GFP glows I13453+RBS+C0072+TT+K091105+E0240 Test Tet and Mnt repression: Some promoter→TetR+Mnt+t+t+pMnt/Tet→GFP does not glows I13453+RBS+C0040+C0072+TT+K091105+E0240 add aTc does not glow

LuxR	cI	AI-1 + GFP	AI-1 - GFP
0	0	0	0
1	0	1	0
0	1	0	0
1	1	0	0