Difference between revisions of "Part:BBa K1949000"
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“We found occasionally that C–A base substitution at nucleotide number 532 in the 5’-UTR region (nucleotide number is according to GenBank Accession No. M30139) diminished this growth inhibition effect (data not shown), allowing easy cloning of the genes of interest into MCS. We do not know why this mutation diminished growth inhibition effect but the mutation did not affect productivity of proteins (data not shown).”<br> | “We found occasionally that C–A base substitution at nucleotide number 532 in the 5’-UTR region (nucleotide number is according to GenBank Accession No. M30139) diminished this growth inhibition effect (data not shown), allowing easy cloning of the genes of interest into MCS. We do not know why this mutation diminished growth inhibition effect but the mutation did not affect productivity of proteins (data not shown).”<br> | ||
| − | Nakashima,N and Tamura,T. 2004. Cell-free protein synthesis using cell extract of <i>Pseudomonas fluorescens</i> and <i>CspA<i> promoter. Biochemical and Biophysical Research Communications 319 (2004) 672 | + | Nakashima,N and Tamura,T. 2004. Cell-free protein synthesis using cell extract of <i>Pseudomonas fluorescens</i> and <i>CspA</i> promoter. Biochemical and Biophysical Research Communications 319 (2004) 672 |
Revision as of 06:07, 10 October 2016
Contents
cold inducible promoter (Pcold)
This new promoter, a cold inducible promoter (we call this Pcold) consists of the cspA promoter, Cold Box, 5’-UTR, RBS and DB. This promoter is used to effectively produce proteins at low temperatures.
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.
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 florescent 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.
Design
We applied a point mutation to the Pcold in order to be easy to clone it. We changed the 178th base C to A. An article which we referred describes as stated below.
“We found occasionally that C–A base substitution at nucleotide number 532 in the 5’-UTR region (nucleotide number is according to GenBank Accession No. M30139) diminished this growth inhibition effect (data not shown), allowing easy cloning of the genes of interest into MCS. We do not know why this mutation diminished growth inhibition effect but the mutation did not affect productivity of proteins (data not shown).”
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 308
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]