Difference between revisions of "Part:BBa J45017:Design"

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<partinfo>BBa_J45017 short</partinfo>
 
<partinfo>BBa_J45017 short</partinfo>
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===Design Notes===
 
===Design Notes===
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*Constructed by PCR
  
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Salicylate is made from chorismate via isochorismate. The reactions are catalysed by PchA (isochorismate synthase) and PchB (isochorismate-pyruvate lyase).  Combined expression of the two proteins from one BioBrick part successfully generates salicylate in ''E. coli.''  For additional design features see [http://parts.mit.edu/wiki/index.php/MIT_2006 the MIT iGEM 2007 wiki].
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====Template====
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pME3366 plasmid
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*We thank Dr. Cornelia Reimmann, Department of Fundamental Microbiology, Universite de Lausanne for kindly providing the pME3366 plasmid encoding the ''pchBA'' genes. Information about Dr. Reimmann's work is available at [http://www.unil.ch/dmf/page16594.html her website].
  
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===Source===
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====Forward primer====
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<code>5'- GTT TCT ACG AAT TCG CGG CCG CTT CTA G'''at gaa aac tcc cga aga ctg cac''' -3'</code>
  
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PchB and PchA are found in ''Pseudomonas aeruginosa''.  The plasmid was kindly sent to the MIT iGEM team by Dr. Cornelia Reimmann from the Universite de Lausanne.  Information about Dr. Reimmann's work is available at [http://www.unil.ch/dmf/page16594.html her website].
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====Reverse primer====
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<code>5'- GTT TCT TTA CTA GTA TTA TT'''a ggc gac gcc gcg ctg''' -3'</code>
  
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===References===
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====Forward mutagenesis primer====
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<code>5'- GAT CGA CCC ATT GGA CCC GCT ACA GGT ATT CGG TGC -3'</code>
  
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L. Serino, C. Reimmann, H. Baur, M. Beyeler, P. Visca & D. Haas. 1995. "Structural genes for salicylate biosynthesis from chorismate in ''Pseudomonas aeruginosa''." ''Mol. Gen. Genet.'' '''249''', 217-228.  This is the original paper describing ''pchBA''.
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====Reverse mutagenesis primer====
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<code>5'- GCA CCG AAT ACC TGT AGC GGG TCC AAT GGG TCG ATC -3'</code>
  
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C. Gaille, C. Reimmann & D. Haas. 2003. "Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of ''Pseudomonas aeruginosa." J. Biol. Chem.'' '''278''', 16893-16898.  Information about the enzyme encoded by ''PchA.''
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===Source===
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''pchBA'' genes from ''Pseudomonas aeruginosa'' (GenBank [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=1628425 X82644])
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===References===
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Original paper describing ''pchBA'' <cite>Serino-MolGenGenet-1995</cite>.  Information about the enzyme encoded by ''PchA'' <cite>Gaille-JBC-2003</cite>.
  
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J. Zaitseva, J. Lu, K.L. Olechoski & A.L. Lamb. 2006. "Two crystal structures of the isochorismate pyruvate lyase from ''Pseudomonas aeruginosa.''" ''J. Biol. Chem.'' '''281,'''33441-9.
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<biblio>
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#Serino-MolGenGenet-1995 pmid=7500944
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#Serino-JBacteriol-1997 pmid=8982005
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#Gaille-JBC-2002 pmid=11937513
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#Gaille-JBC-2003 pmid=12624097
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#Zaitseva-JBC-2006 pmid=16914555
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</biblio>

Latest revision as of 22:30, 7 March 2008

isochorismate pyruvate-lyase and isochorismate synthase (pchBA); converts chorismate to salicylate


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 307
    Illegal NgoMIV site found at 711
    Illegal NgoMIV site found at 836
    Illegal NgoMIV site found at 847
    Illegal NgoMIV site found at 1128
    Illegal NgoMIV site found at 1596
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

  • Constructed by PCR

Template

pME3366 plasmid

  • We thank Dr. Cornelia Reimmann, Department of Fundamental Microbiology, Universite de Lausanne for kindly providing the pME3366 plasmid encoding the pchBA genes. Information about Dr. Reimmann's work is available at [http://www.unil.ch/dmf/page16594.html her website].

Forward primer

5'- GTT TCT ACG AAT TCG CGG CCG CTT CTA Gat gaa aac tcc cga aga ctg cac -3'

Reverse primer

5'- GTT TCT TTA CTA GTA TTA TTa ggc gac gcc gcg ctg -3'

Forward mutagenesis primer

5'- GAT CGA CCC ATT GGA CCC GCT ACA GGT ATT CGG TGC -3'

Reverse mutagenesis primer

5'- GCA CCG AAT ACC TGT AGC GGG TCC AAT GGG TCG ATC -3'

Source

pchBA genes from Pseudomonas aeruginosa (GenBank [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=1628425 X82644])

References

Original paper describing pchBA Serino-MolGenGenet-1995. Information about the enzyme encoded by PchA Gaille-JBC-2003.

<biblio>

  1. Serino-MolGenGenet-1995 pmid=7500944
  2. Serino-JBacteriol-1997 pmid=8982005
  3. Gaille-JBC-2002 pmid=11937513
  4. Gaille-JBC-2003 pmid=12624097
  5. Zaitseva-JBC-2006 pmid=16914555

</biblio>