Difference between revisions of "Part:BBa K557001:Design"

Revision as of 14:17, 2 October 2011 (view source) Pythonwawa (Talk | contribs) (→‎Design Notes) ← Older edit		Latest revision as of 17:55, 2 October 2011 (view source) Pythonwawa (Talk | contribs) (→‎References)
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	===Source===		===Source===
−	Escherichia coli, the strain has always been kept in the lab of USTC-IGEM	+	''Escherichia coli'', the strain has always been kept in the lab of USTC-IGEM
	===References===		===References===
		+	Shana Topp, Justin P. Gallivan (2006) Guiding Bacteria with Small Molecules and RNA. J.Am.Chem.Soc, 129,6870-6811

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Latest revision as of 17:55, 2 October 2011

Aptamer-CheZ: theophylline-sensitive synthetic riboswitch+phosphatase which dephosphorylates CheY-P

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:

Besides, we used special protocal of PCR:
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