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From 2011.igem.org

• Aptamer-cheZ Device Construction

The cheZ gene was cloned from E.coli TOP10 strain using PCR. A cassete containing a theophylline-sensitive aptamer, and the cheZ gene was assembled using PCR and was subcloned into the SpeI and PstI sites of pSB1A2 containing the lac promoter.

Clone Primer:

Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGATGCAACCATCAATCAAACC-3' Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGTCTATCCAACAAATCGT-3'

Assemble Primer:

Forward:5'-GTTTCGAATTCGCGGCCGCTTCTAGGGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACCCCGCTGCAAGACAACAAG ATGCAACCATCAATCAAACC-3'

Reverse:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGACTATCCAACAAATCGT-3'

Assemble condition: 96℃ 10min, (96℃ 30s, 56℃ 30s, 72℃ 1min)16 cycles and each cycle the annealing temperature increase 1℃, (96℃ 30s, 72℃ 30s, 72℃ 1min)20 cycles,72℃ 10min and holding at 4℃.