In order to illuminate the construction of our AND gate more clearly, we further decoupled the AND gate into an input sensor module and a core module:
The sensor module includes two inducible promoters. We chose two promoters which can be induced by specific environmental small molecules: one is a salicylate-activated promoter Psal(BBa_K228004) to control the supD gene, and the other is an arabinose-activated promoter PBAD (BBa_I13453) to control the T7ptag gene. Both of the inducible promoters can only work in the presence of corresponding activators, so we cloned all the activators downstream of a constitutive promoter (BBa_I14033) to make protein generators. Then the generators are placed upstream of the corresponding inducible promoters. In particular, the araC protein (BBa_K228008) is expressed upstream of PBad to make a arabinose sensor which can express downstream gene when induced by arabinose. However, the salicylate sensor is originally on the pSal-SupD plasmid which Voigt Lab mailed us instead of building from standard parts. We found the sequence from NCBI and ensured that there are no standard enzymes cleavage sites inside of its sequence. After the preparation work, we designed primers and PCR it from the plasmid.
SupD gene and T7ptag gene constitute the core of the AND Gate. Firstly, we use PCR to adapt T7ptag (BBa_K228000) and supD (BBa_K228001) into standard biobrick. The primer is designed according to the paper "Environmental signal integration by a modular AND Gate", we maintained the complementary sequence in the original primer, and replaced the enzyme cleavage site in the original primer with standard prefix and suffix. Secondly, we placed a strong terminator BBa_B0015 downstream of the two parts to prevent these parts from interfering with each other. In addition, for T7ptag, we clone it downstream of 9 different RBSs. For supD, as it just codes tRNA, no RBS is needed. Thus, the complete core of AND gate is constructed. Finally, aforesaid standard regulatory parts were placed upstream of the T7ptag and supD generators to provide this device.