Difference between revisions of "Part:BBa K1949001"

Revision as of 10:42, 10 October 2016

Pcold-gfp

Fig. 1. We characterize cold inducible promoter (Pcold) by this construction.

This promoter is used to effectively produce proteins at low temperatures. This new promoter, a cold inducible promoter (we call this Pcold) consists of the cspA promoter, Cold Box, 5’-UTR, RBS and DB. The combination (of cspA promoter which is active at both low and high temperature, Cold box which inhibits excessive gene expression, 5’UTR which is stable at only low temperature, and DB which function as an extra RBS) activates gene expression at low temperature.

Characterization

We measured florescence intensity per turbidity using cells cultured at 18℃ and 37℃ to confirm function of Pcold. The cells had a plasmid which carries Pcold-gfp or Ptet-gfp.

E. coli cells which carry the Ptet-RBS-gfp plasmid was cultured at 18℃, and florescence intensity was measured at indicated time points. Also, the same experiment was performed at 37℃. We thought this was because gfp is easy to fold for florescence at low temperature. By contrast, florescence of E. coli which included Ptet-RBS-gfp at 18℃ was about eight times as florescence as it at 37℃. By this result, we confirm Pcold activates gene expression at low temperature.

Biobrick Tips

This part is not able to be used for most common assembly, because restriction enzyme digestion with XbaI and SpeI generates an unexpected stop codon. Therefore, this part do not meet the criteria of basic parts construction. Our team generated a unique digestion site, BamHI at the upstream of the suffix. We recommend to use this BamHI site for cloning.

Sequence and Features