Difference between revisions of "Part:BBa K1949000"
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<span style="margin-left: 10px;"><i>E. coli</i> cells which carry the P<i>tet</i>-RBS-<i>gfp</i> 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 intensity of <i>E. coli</i> which included P<i>tet</i>-RBS-<i>gfp</i> at 18℃ was about eight times as florescence as it at 37℃. By this result, we confirm P<i>cold</i> activates gene expression at low temperature.
 
<span style="margin-left: 10px;"><i>E. coli</i> cells which carry the P<i>tet</i>-RBS-<i>gfp</i> 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 intensity of <i>E. coli</i> which included P<i>tet</i>-RBS-<i>gfp</i> at 18℃ was about eight times as florescence as it at 37℃. By this result, we confirm P<i>cold</i> activates gene expression at low temperature.
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===Biobrick Tips===
 
===Biobrick Tips===
 
<span style="margin-left: 10px;">This part is not able to be used for most common assembly, because restriction enzyme digestion with <i>Xba</i>I and <i>Spe</i>I generates an unexpected stop codon. Therefore, this part do not meet the criteria of basic parts construction. Our team generated a unique digestion site, <i>Bam</i>HI at the upstream of the suffix. We recommend to use this <i>Bam</i>HI site for cloning.
 
<span style="margin-left: 10px;">This part is not able to be used for most common assembly, because restriction enzyme digestion with <i>Xba</i>I and <i>Spe</i>I generates an unexpected stop codon. Therefore, this part do not meet the criteria of basic parts construction. Our team generated a unique digestion site, <i>Bam</i>HI at the upstream of the suffix. We recommend to use this <i>Bam</i>HI site for cloning.
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 +
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===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.
 +
 +
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.
 +
 +
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
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Revision as of 19:57, 11 October 2016

cold inducible promoter (Pcold)

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

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


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.

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.

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 308
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]