Part:BBa_K557001:Design
Aptamer-CheZ: theophylline-sensitive synthetic riboswitch+phosphatase which dephosphorylates CheY-P
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We found the sequence of this part in literature and tried to design primers to construct it. But we were faced with a challenging problem in primer design, which comes from the basic properties of the aptamer involved. The aptamer tends to pair the ribosome binding site in the absence of theophylline, which is true in PCR, and enables primers to pair to the corresponding parts and the new strand to elongate. We made contact with the author of our reference and found they took different strategies in assembling the aptamer to CheZ instead of PCR. But thanks to the warm help of Professor J. S. Parkinson and Professor J. P. Gallivan, we acquired the sequence of primers used in assembling the aptamer to another gene and designed our own after analyzing them:
reverse primer:5'-GTTTCCTGCAGCGGCCGCTACTAGTATTATTAAAATCCAAGACTATCCAACAAATCGT-3'
96℃ 10min;
96℃ 30s, 56℃ 30s, 72℃ 1min for 16 cycles and each cycle the annealing temperature increases by 1℃;
96℃ 30s, 72℃ 30s, 72℃ 1min for 20 cycles;
72℃ 10min.
Source
Escherichia coli, the strain has always been kept in the lab of USTC-IGEM
References
Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811