Difference between revisions of "Part:BBa K1949001"

Revision as of 20:26, 18 October 2016

Pcold-gfp

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 temperatures.

Characterization

RFU (Relative Fluorescence Units) of GFP per turbidity was measured using cells cultured at 18℃ and 37℃ to confirm function of Pcold. The cells harbored a plasmid which carries Pcold-gfp or Ptet-rbs-gfp.

Fig.1 RFU of E. coli which harbored Ptet-rbs-gfp at 18℃ was about twice higher than that at 37℃.

Fig.2 RFU of E. coli which harbored Pcold-rbs-gfp at 18℃ was about eight fold higher than that at 37℃.

E. coli cells which carry the Ptet-rbs-gfp plasmid was cultured at 18℃, and RFU of GFP was measured at indicated time points. (Fig.1)Also, the same experiment was performed at 37℃. We thought this result was obtained because GFP is easily folded into correct structures at low temperatures. By contrast, RFU of E. coli which harbored Pcold-rbs-gfp at 18℃ was about eight fold higher than that at 37℃. From this result, we confirmed Pcold activates gene expression at low temperatures.(Fig.2)

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.

Reference

Famg L,Hou Y and Inoue M.1998. Role of Escherichia coli cspA promoter sequences and adaptation of translational apparatus in the cold shock response. Mol Gen Genet. 1997 Oct;256(3):282-90

Goldenberg D,Azar I,Oppenheim AB,Brandi A,Pon CL,Gualerzi CO. Role of the cold-box region in the 5' untranslated region of the cspA mRNA in its transient expression at low temperature in Escherichia coli. J Bacteriol. 1998 Jan;180(1):90-5.

Nakashima N and Tamura T. 2004. Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter. Biochemical and Biophysical Research Communications 319 (2004) 672

Yamanaka K.1999. Cold Shock Response in Escherichia coli. J. Mol. Microbiol. Biotechnol. (1999) 1(2): 193-202

Sequence and Features