Cloning and Characterization of an AND Gate Genetic Circuit

The Luria-Bertani (LB) media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) is obtained from Fisher Scientific (Pittsburgh, PA). The supplemented minimal media contains M9 minimal salts (6.8 g/L Na₂PO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl) from Sigma, 2 mM MgSO4 (Fischer Scientific), 100 μM CaCl₂ (Fischer Scientific), 0.4% glucose (Sigma), 0.05 g/L leucine (Acros Organics, Belgium), 5 μg/mL chloramphenicol (Acros Organics), and an adjusted pH of 7.4. The expression system is a ColE1 vector with chloramphenicol resistance (derived from pProTet, Clontech). The expression cassette contains a σ⁷⁰ constitutive promoter (BioBrick J23100), the RBS sequence, followed by the mRFP1 fluorescent protein reporter. XbaI and SacI restriction sites are located before the RBS and after the start codon. An RBS with a desired sequence is inserted into the expression vector using standard cloning techniques. Pairs of complementary oligonucleotides are designed with XbaI and SacI overhangs and the vector is digested with XbaI and SacI restriction enzymes (NEB, Ipswich, MA). Ligation of the annealed oligonucleotides with cut vector results in a nicked plasmid, which is transformed into E. coli DH10B cells. Sequencing is used to verify a correct clone.
The AND gate genetic circuit is composed of three plasmids: pBACr-AraT7940, pBR939b, and pAC-SalSer914 with kanamycin, ampicillin, and chloramphenicol resistance markers, respectively. The P_BAD promoter maximum expression level was modified by inserting designed synthetic RBSs on plasmid pBACr-AraT7940. Plasmid pBACr-AraT7940 was digested with BamHI and ApaLI enzymes and pairs of oligonucleotides were designed to contain the desired RBS sequence and corresponding overhangs. Ligation, transformation, selection, and sequencing proceeded as described above.