Part:BBa_K328001

cspA Promoter + Cold Box + UTR + SD + DB

This is the complete promoter cspA promoter + Cold Box + UTR + SD + DB extracted from E coli DH5Alpha for test about the cold shock response and the ability to control a genetic circuit using it as a cold sensor, is intended to produce proteins after a cold shift and allows us to sintetize thermo-labile proteins also.

CspA Promoter: The promoter consists of 50 bases. This promoter is active at both 37ºC and 15ºC.

Cold box: Is an important region of the 5’ UTR region, which may form a stable stem-loop structure and is implicated in the auto regulation of the cspA system by the HNS gene of E coli.

5’ UTR: This unusual UTR region make the cspA mRNA unstable at 37º C with an average half-life of less than 12 seconds, but more stable at 15°C with an average half-life of 20 minutes and improve the translation at low temperature. In here there is an RNAse cleavage site immediately upstream the Shine-Dalgarno sequence that is considered to be responsible for this unstable messenger stage.

Shine-Dalgarno (SD) sequence: Is common for many messengers is identified as a ribosomal binding site

Downstream Box: Is a fourteen nucleotide sequence that is in the mRNA and is complementary to a region found in the 16S rRNA named as anti-DB sequence , which gives the feature of good translation at low temperature because it function as an extra messenger ribosome binding site. In this way the ribosome can anchor the SD sequence and the DB sequence as a two ribosome-binding site to permit a great cleavage between the ribosome and the messenger

Biobrick Tips:

• It has been observed that when expressing a coding device the synthesis of rna and protein in cold shock will significantly change.
• The effect of cold shock can be observed only in a fast downshift from 37ºC to 10-15ºC
• After cold shock there are two phases. The aclimatation phase where all the Cold inducible proteins, like a gene in control with this biobrick, are highly expressed; this phase takes around 2 hours with the CSPA protein. Later there are an cold adapted phase where the cell proteins recover a better rate of synthesis and all the genes with the cold shock response system become repressed transcriptionally.

PART IMPROVEMENT - IONIS_PARIS 2017

This part was improved upon as https://parts.igem.org/Part:BBa_K2282011 where it was used for conditional expression of an amilCP reporter only at low temperature (~15 C).

References

• Baneyx, François and Mujacic, Mirna; (2002); Cold-Inducible Promoters for Heterologous Protein Expression; from Methods in Molecular Biology; Vol. 205 Humana Press Inc.

• Horn G., et al.; (2007); Structure and function of bacterial cold shock proteins; Cellular and Molecular Life Sciences; Vol. 64 p. 1457 – 1470

• Thieringer A. Heather, Pamela G. Jones, and Masayori Inouye; (1998); Cold Shock and Adaptation; Bio Essays 20.1: 40-57

• Yamanaka, Kunitoshi; (1999); Cold Shock Response in Escherichia coli; Journal of Molecular and Microbiological Biotechnology 1(2): 193-202

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]