Difference between revisions of "Part:BBa K1065106"

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This is the complete wintergreen odor device from iGEM UniTN-Trento 2013 which encodes for four enzymes: PchB, PchA, BSMT1 and SAM.
 
This is the complete wintergreen odor device from iGEM UniTN-Trento 2013 which encodes for four enzymes: PchB, PchA, BSMT1 and SAM.
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The device produces methyl salicylate (a fruit ripening inhibitor) and was used for the development of the project <i>B. Fruity</i> by UniTN Trento 2013.  
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The device produces methyl salicylate (a fruit ripening inhibitor) <html><a href="#ref1" id="ret_ref1">[1]</a></html> and was used for the development of the project <i>B. Fruity</i> by UniTN Trento 2013.  
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This part, containing SAM enzyme <partinfo>BBa_K1065105</partinfo>, is an improvement of part <partinfo>BBa_K1065102</partinfo>.  
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This part, containing SAM enzyme <partinfo>BBa_K1065105</partinfo> <html><a href="#ref2" id="ret_ref2">[2]</a></html>, is an improvement of part <partinfo>BBa_K1065102</partinfo>.  
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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The chorismate, a metabolic intermediate of the Shikimate pathway which many plants and bacteria (like E.coli and B.subtilis ) have, undergoes firstly a reaction of isomerization by the isochorismate synthase, PchA . Salicylate is then obtained by the action of PchB an isochorismate pyruvate lyase. Both enzymes are from the micro-organism Pseudomonas aeruginosa . In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase.
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The chorismate, a metabolic intermediate of the Shikimate pathway present in many plants and bacteria, undergoes at first a reaction of isomerization by the isochorismate synthase, PchA <html><a href="#ref3" id="ret_ref3">[3]</a></html>. Salicylate is then obtained by the action of PchB, an isochorismate pyruvate lyase. Both enzymes are from the microorganism <html><i>Pseudomonas aeruginosa</i> <a href="#ref4" id="ret_ref4">[4]</a></html>. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase.
  
 
<html>
 
<html>
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</html>
 
</html>
  
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===Characterization===
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<html>
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<h3>MeSA detection</h3>
  
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NEB10β cells transformed with Bba_K1065106, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 0.9 ppm for Bba_K1065106 (in the presence of salicylic acid). Non induced cells did not produce any MeSA.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1065106 SequenceAndFeatures</partinfo>
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<center><img style="width:800px;"src="https://static.igem.org/mediawiki/parts/e/e4/Unitn2013-BBa_K1065106_inducedSA.png"></center>
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<center><p style="width:600px; margin-bottom:60px; text-align:justify"><b>Figure 1</b> Quantification of MeSA by gas chromatography. a)NEB10β cells transformed with Bba_K1065106 and induced with 5mM arabinose and supplemented with salycilic acid, b) 15 ppm reference point.
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</p></center> </html>
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This analysis was performed also with the <partinfo>BBa_K1065102</partinfo> and in that case more MeSA was detected, in particular in liquid phase.
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Please note that we received the DNA sequencing results of the MIT part (<partinfo>BBa_J45300</partinfo>) and of our complete device (built with MIT parts). We realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.
  
  
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<partinfo>BBa_K1065106 parameters</partinfo>
 
<partinfo>BBa_K1065106 parameters</partinfo>
 
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===References===
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<html><ol>
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<li><a id="ref1"></a>Chang-Kui Ding, Chien Yi Wang (2003). The dual effects of methyl salicylate on ripening and expression of ethylene biosynthetic genes in tomato fruit, Plant Science Volume 164, Pages 589–596.</li>
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<li><a id="ref2"></a> Wang et al., (2002) Ethylene biosynthesis and signaling networks; Ecker J. R. Plant Cell , Supplement, S131-S151</li>
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<li><a id="ref3"></a> Marianne C. Verberne at al., (1999). Salicylic acid biosynthesis, Biochemistry and Molecular Biology of Plant Hormones, 295–312. <br></li>
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<li><a id="ref4"></a>Florence Negre at al., (2002) Novel S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers, Archives of Biochemistry and Biophysics, Volume 406, Issue 2, Pages 261–270.</li>
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</html>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1065106 SequenceAndFeatures</partinfo>

Latest revision as of 23:16, 4 October 2013

Wintergreen device with SAM: Plac + PCHBA + araCpBAD+ BSMT1 + Plac + SAM synthetase

This is the complete wintergreen odor device from iGEM UniTN-Trento 2013 which encodes for four enzymes: PchB, PchA, BSMT1 and SAM. The device produces methyl salicylate (a fruit ripening inhibitor) [1] and was used for the development of the project B. Fruity by UniTN Trento 2013. This part, containing SAM enzyme BBa_K1065105 [2], is an improvement of part BBa_K1065102.

Usage and Biology

The chorismate, a metabolic intermediate of the Shikimate pathway present in many plants and bacteria, undergoes at first a reaction of isomerization by the isochorismate synthase, PchA [3]. Salicylate is then obtained by the action of PchB, an isochorismate pyruvate lyase. Both enzymes are from the microorganism Pseudomonas aeruginosa [4]. In the final part of the reaction, BSMT1, a methyltransferase, transfers a methyl group from the S-adenosyl-L-methionine synthesized by the SAM synthetase.

Characterization

MeSA detection

NEB10β cells transformed with Bba_K1065106, were grown in M9 medium, induced with 5 mM arabinose and in some cases supplemented with 2mM of salicylic acid. After 4 hours we performed the gas chromatographyc analyis with a column optimized for the fast analysis of volatile compounds (J&W GC Column Performance Summary-Agilent Tecnologies). Peak corresponding to MeSA eluted at a ritension time of 5.5 min. The quantitative analysis done by integration of the peak area showed that small amounts of MeSA are released in the gas phase under this experimental condition: 0.9 ppm for Bba_K1065106 (in the presence of salicylic acid). Non induced cells did not produce any MeSA.

Figure 1 Quantification of MeSA by gas chromatography. a)NEB10β cells transformed with Bba_K1065106 and induced with 5mM arabinose and supplemented with salycilic acid, b) 15 ppm reference point.

This analysis was performed also with the BBa_K1065102 and in that case more MeSA was detected, in particular in liquid phase.

Please note that we received the DNA sequencing results of the MIT part (BBa_J45300) and of our complete device (built with MIT parts). We realised that the pLAC promoter was missing the -35 box, thus generating a less strong promoter. We believe that this problem can significantly affect the correct functioning of the device. We are now in the process of improving this part by mutagenesis to rebuild a full functional pLAC promoter.


References

  1. Chang-Kui Ding, Chien Yi Wang (2003). The dual effects of methyl salicylate on ripening and expression of ethylene biosynthetic genes in tomato fruit, Plant Science Volume 164, Pages 589–596.
  2. Wang et al., (2002) Ethylene biosynthesis and signaling networks; Ecker J. R. Plant Cell , Supplement, S131-S151
  3. Marianne C. Verberne at al., (1999). Salicylic acid biosynthesis, Biochemistry and Molecular Biology of Plant Hormones, 295–312.
  4. Florence Negre at al., (2002) Novel S-adenosyl-l-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers, Archives of Biochemistry and Biophysics, Volume 406, Issue 2, Pages 261–270.


    5. 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 3107
      Illegal BamHI site found at 3856
      Illegal BamHI site found at 4736
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal NgoMIV site found at 389
      Illegal NgoMIV site found at 793
      Illegal NgoMIV site found at 918
      Illegal NgoMIV site found at 929
      Illegal NgoMIV site found at 1210
      Illegal NgoMIV site found at 1678
      Illegal AgeI site found at 2942
      Illegal AgeI site found at 5316
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI site found at 3397
      Illegal SapI site found at 2924