Difference between revisions of "Part:BBa K1949000:Experience"
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===Materials and Methods===
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===More information===
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<i>cspA</i> Promoter is active at both 37℃ and 15℃ invariably. This promoter consists of 17 genes.
  
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====Construction====
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Cold box is an important region of 5’UTR region and This consists of 17 genes. This region may form stable stem-loop structure. It traps RNA polymerase and inhibits  　excessive gene expression.
  
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-Strain
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5’UTR is unusual UTR. At 37℃, this makes cspA mRNA unstable with its average half-life 12 seconds. But at 15℃, this makes cspA mRNA relatively stable with its average half-life 20 minutes. So, gene expression at low temperature is activated.
  
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All the sample were BL21(DE3) strain
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RBS sequence is also known as SD (Shine‐Dalgarno sequence)
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-Plasmids
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-Pcold-gfp (pSB1C3)
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-Positive Control: Pcon-rbs-gfp (pSB1C3)
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-Negative Control: empty vector (pSB1C3)
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====Assay Protocol====
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1. Prepare overnight cultures for each sample in 3 mL LB medium containing chloramphenicol (34 microg / mL) at 37 ºC for 12h.
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2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the OD600 becomes around 0.05 in triplicate (fresh culture).
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3. Incubate the triplicated fresh cultures each at 28ºC so that the OD600 reaches 0.3 to 0.4
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4. Incubate the triplicated fresh cultures each in water and ice bath for 2min.
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5. Incubate the triplicated fresh cultures each at 18ºC, and 37ºC for each sample for 3 h.
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6. Measure the OD and the fluorescence intensity.
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===Reference===
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[1] Nakashima N and Tamura T. Cell-free protein synthesis using cell extract of Pseudomonas fluorescence and CspA promoter. Biochemical and Biophysical Research Communications. Biochem Biophys Res Commun. 2004 Jun 25;319(2):671-6.
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Downstream Box(DB) consists of 14 genes and this is complementary to anti-DB sequence found in 16S rRNA. This plays role as an extra RBS
  
 
===Applications of BBa_K1949000===
 
===Applications of BBa_K1949000===

Revision as of 05:01, 16 October 2016


More information

cspA Promoter is active at both 37℃ and 15℃ invariably. This promoter consists of 17 genes.

Cold box is an important region of 5’UTR region and This consists of 17 genes. This region may form stable stem-loop structure. It traps RNA polymerase and inhibits 　excessive gene expression.

5’UTR is unusual UTR. At 37℃, this makes cspA mRNA unstable with its average half-life 12 seconds. But at 15℃, this makes cspA mRNA relatively stable with its average half-life 20 minutes. So, gene expression at low temperature is activated.

RBS sequence is also known as SD (Shine‐Dalgarno sequence)

Downstream Box(DB) consists of 14 genes and this is complementary to anti-DB sequence found in 16S rRNA. This plays role as an extra RBS

Applications of BBa_K1949000

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