Difference between revisions of "Part:BBa J45992:Design"

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<partinfo>BBa_J45992 short</partinfo>
 
<partinfo>BBa_J45992 short</partinfo>
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===Design Notes===
 
===Design Notes===
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Primers used to PCR this part out of the ''E.coli'' genome:  
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*We considered different designs for stationary phase regulation of odor production.  All designs focused on using transcriptional control devices to regulate the odor enzyme generators and thus odor production.  One candidate design for stationary phase regulation was to use a transcriptional promoter that is only active in stationary phase.  Several ''E. coli'' promoters that are primarily active in stationary phase have been previously characterized <cite>Schellhorn-1998, Vijayakumar-2004</cite>.  Thus, we could evaluate various stationary phase promoters as potential growth phase dependent transcriptional control devices.
 +
*We evaluated three stationary phase promoters as potential stationary phase dependent transcriptional control devices.  We first tested the promoter that controls transcription of ''rpoS'' in ''E. coli'' <cite>Hengge-Aronis-1993</cite>.  The ''rpoS'' gene encodes &sigma;<sup>S</sup> factor, a transcription factor known to be present at higher levels during late exponential phase and early stationary phase <cite>Lange, Tanaka</cite>.  Our preliminary experiments with a plasmid encoding the ''rpoS'' promoter controlling transcription of green fluorescent protein (GFP, plasmid pBS-rrnBTrpoSpUV) demonstrated that the ''rpoS'' promoter did not produce significantly higher fluorescence in stationary phase than during exponential growth <cite>Funabashi</cite>.  We then identified a second ''E. coli'' stationary phase promoter for evaluation: the promoter that controls transcription of ''osmY'' <cite>Yim-1992, Yim-1994</cite>.  Expression of ''osmY'' is dependent on &sigma;<sup>S</sup> ''in vivo'' <cite>Hengge-Aronis-1993</cite>.  Schellhorn ''et al.'' previously demonstrated that an \textit{osmY::lacZ} fusion generated the highest transcriptional signal in stationary phase as compared to nine other &sigma;<sup>S</sup>-dependent promoter-''lacZ'' fusions <cite>Schellhorn-1998, Vijayakumar-2004</cite>.  In addition, the ''osmY::lacZ'' fusion generated only a small transcriptional signal during exponential growth. 
 +
*Constructed by PCR
 +
 
 +
====Template====
 +
''Escherichia coli'' strain MG1655
  
 
====Forward Primer====
 
====Forward Primer====
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*5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCTG GCA CAG GAA CGT TAT C - 3'
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<code>5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCT GGC ACA GGA ACG TTA TC - 3'</code>
−
*Total Length: 47 bp
+
−
*Annealing Length: 19 bp
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−
*GC Content: 52.6%
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−
*Melting Temperature: 53.4 degrees C
+
−
*hairpin deltaG: -3.99 kcal/mol
+
−
*self dimer deltaG: -98.3 kcal/mol
+
  
 
====Reverse Primer====
 
====Reverse Primer====
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*5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG- 3'
+
<code>5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG AAA CCG- 3'</code>
−
*Total Length: 54 bp
+
−
*Annealing Length: 25 bp
+
−
*GC Content: 20.0%
+
−
*Melting Temperature: 46.4 degrees C
+
−
*hairpin deltaG: -2.86 kcal/mol
+
−
*self dimer deltaG: -99.44 kcal/mol
+
−
*heterodimer deltaG with Forward Primer: -99.44 kcal/mol
+
  
 
===Source===
 
===Source===
−
 
+
*Promoter controlling expression of ''osmY'' from ''Escherichia coli'' (GenBank [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=453110 S67278])
−
This part was PCRed out of E. coli's genome with the primers listed in the design notes section.
+
  
 
===References===
 
===References===
 +
<biblio>
 +
#Yim-1994 pmid=8282684
 +
#Schellhorn-1998 pmid=9829938
 +
#Wise pmid=8631665
 +
#Funabashi pmid=11879714
 +
#Makinoshima pmid=11985708
 +
#Yim-1992 pmid=1317380
 +
#Hengge-Aronis-1993 pmid=8416901
 +
#Vijaykumar-2004 pmid=15576800
 +
#Tanaka pmid=8475100
 +
#Lange pmid=1849609
 +
</biblio>
  
  
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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Nucleotide&list_uids=453110&dopt=GenBank
 
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http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=8282684
 
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http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=9829938
 
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http://www.blackwell-synergy.com/doi/abs/10.1046/j.1365-2958.2002.02746.x
 
−
 
−
http://www.pubmedcentral.org/articlerender.fcgi?artid=178012
 
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http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-456T3CY-3&_coverDate=04%2F25%2F2002&_alid=423325879&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=4943&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=7d6f4bac2242f1baa6b2b15260904531
 
  
 
Neidhart, Frederick C. ''Escherichia coli and Salmonella''. Washington: ASM Press, 1996.
 
Neidhart, Frederick C. ''Escherichia coli and Salmonella''. Washington: ASM Press, 1996.

Latest revision as of 01:24, 9 March 2008

Full-length stationary phase osmY promoter


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]


Design Notes

  • We considered different designs for stationary phase regulation of odor production. All designs focused on using transcriptional control devices to regulate the odor enzyme generators and thus odor production. One candidate design for stationary phase regulation was to use a transcriptional promoter that is only active in stationary phase. Several E. coli promoters that are primarily active in stationary phase have been previously characterized Schellhorn-1998, Vijayakumar-2004. Thus, we could evaluate various stationary phase promoters as potential growth phase dependent transcriptional control devices.
  • We evaluated three stationary phase promoters as potential stationary phase dependent transcriptional control devices. We first tested the promoter that controls transcription of rpoS in E. coli Hengge-Aronis-1993. The rpoS gene encodes σS factor, a transcription factor known to be present at higher levels during late exponential phase and early stationary phase Lange, Tanaka. Our preliminary experiments with a plasmid encoding the rpoS promoter controlling transcription of green fluorescent protein (GFP, plasmid pBS-rrnBTrpoSpUV) demonstrated that the rpoS promoter did not produce significantly higher fluorescence in stationary phase than during exponential growth Funabashi. We then identified a second E. coli stationary phase promoter for evaluation: the promoter that controls transcription of osmY Yim-1992, Yim-1994. Expression of osmY is dependent on σS in vivo Hengge-Aronis-1993. Schellhorn et al. previously demonstrated that an \textit{osmY::lacZ} fusion generated the highest transcriptional signal in stationary phase as compared to nine other σS-dependent promoter-lacZ fusions Schellhorn-1998, Vijayakumar-2004. In addition, the osmY::lacZ fusion generated only a small transcriptional signal during exponential growth.
  • Constructed by PCR

Template

Escherichia coli strain MG1655

Forward Primer

5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCT GGC ACA GGA ACG TTA TC - 3'

Reverse Primer

5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG AAA CCG- 3'

Source

  • Promoter controlling expression of osmY from Escherichia coli (GenBank [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=453110 S67278])

References

<biblio>

  1. Yim-1994 pmid=8282684
  2. Schellhorn-1998 pmid=9829938
  3. Wise pmid=8631665
  4. Funabashi pmid=11879714
  5. Makinoshima pmid=11985708
  6. Yim-1992 pmid=1317380
  7. Hengge-Aronis-1993 pmid=8416901
  8. Vijaykumar-2004 pmid=15576800
  9. Tanaka pmid=8475100
  10. Lange pmid=1849609

</biblio>


Neidhart, Frederick C. Escherichia coli and Salmonella. Washington: ASM Press, 1996.