Difference between revisions of "Part:BBa K801077:Design"

(→‎Design Notes)
 
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<br>'''Keywords:'''
 
<br>'''Keywords:'''
 
<!--These keywords are necessary to find your part using a fulltext sarch.-->
 
<!--These keywords are necessary to find your part using a fulltext sarch.-->
−
<!--keyword_1, keyword_2, keyword_3, keyword_4, keyword_5-->
+
caffeine, TEF1, TEF2, ADH1, expression cassette
  
 
<br>'''Abbreviations:'''
 
<br>'''Abbreviations:'''
 
<!--*used_abbreviation_1 = full_name_of_used_abbreviations_1-->
 
<!--*used_abbreviation_1 = full_name_of_used_abbreviations_1-->
 
<!--*used_abbreviation_2 = full_name_of_used_abbreviations_2-->
 
<!--*used_abbreviation_2 = full_name_of_used_abbreviations_2-->
 +
* CaXMT1 = coffea arabica xanthosine N-methyltransferase 1
 +
 +
* CaMXMT1 = coffea arabica 7-methylxanthine N-methyltransferase 1
 +
 +
* CaDXMT1 = coffea arabica 3,7-dimethylxanthine N-methyltransferase 1
  
 
===Design Notes===
 
===Design Notes===
  
 
'''Related BioBrick:'''
 
'''Related BioBrick:'''
−
<!--*Other versions:[https://parts.igem.org/wiki/index.php?title=Part:BBa_?????? BBa_??????: Name_of_part] -->
+
[https://parts.igem.org/Part:BBa_K801073 BBa_K801073]
−
<!--*Related BioBricks:[https://parts.igem.org/wiki/index.php?title=Part:BBa_?????? BBa_??????: Name_of_part]
+
[https://parts.igem.org/Part:BBa_K801073 BBa_K801074]
 +
[https://parts.igem.org/Part:BBa_K801076 BBa_K801075]
 +
[https://parts.igem.org/Part:BBa_K801077 BBa_K801076]
 +
<!--*Other versions:[https://parts.igem.org/wiki/index.php?title=Part:BBa_K801076: Name_of_part] -->  
 +
<!--*Related BioBricks:[https://parts.igem.org/wiki/index.php?title=Part:BBa_K801074 BBa_K801075: Name_of_part]
  
 
'''Cloning details:'''<br>
 
'''Cloning details:'''<br>
 +
* cloning in RFC10
 
<!--*Designed in RFC10/RFC23/RFC25/RFC25_N-part-->
 
<!--*Designed in RFC10/RFC23/RFC25/RFC25_N-part-->
 
<!--*Mutation C889G to delete XbaI restriction site-->
 
<!--*Mutation C889G to delete XbaI restriction site-->
Line 27: Line 37:
  
 
'''Quality control measures:'''<br>
 
'''Quality control measures:'''<br>
 +
Test digest has been performed with use of Xba1 and Pst1 (see mainpage)
 +
Part hasn´t been sequenced
 
<!--*Test digestion using ?enzyme1? & ?enzyme2?/Not yet performed-->
 
<!--*Test digestion using ?enzyme1? & ?enzyme2?/Not yet performed-->
 
<!--*Sequencing using primer ?primer_name?/Not yet sequenced-->
 
<!--*Sequencing using primer ?primer_name?/Not yet sequenced-->
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'''Backbone:'''<br>
 
'''Backbone:'''<br>
−
<!--*Backbone name: pSB1C3'/?backbone_name?-->
+
*Backbone name: pSB1C3'
 +
* Resistance: Cp
 
<!--*Resistance: Amp/Cp/Kan/Tet-->
 
<!--*Resistance: Amp/Cp/Kan/Tet-->
 
<!--*Copynumber: low/medium/high-->
 
<!--*Copynumber: low/medium/high-->
  
 
'''Protein coding:'''<br>
 
'''Protein coding:'''<br>
 +
* Protein: CaXMT1-Strep-tag, CaMXTM1-Strep-tag, CaDXMT1-Strep-tag
 
<!--*Protein: ?Name_of_gene_product? [Nucleotide 1 to ???]-->
 
<!--*Protein: ?Name_of_gene_product? [Nucleotide 1 to ???]-->
 
<!--*The protein has the amino acid replacements ???99??? to ???99???.-->
 
<!--*The protein has the amino acid replacements ???99??? to ???99???.-->
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'''Enzymatic activity:'''
 
'''Enzymatic activity:'''
 +
*see above related biobricks
 
<!--none/EC-number ?.?.?.?-->
 
<!--none/EC-number ?.?.?.?-->
  
 
'''Cytotoxicity:'''<br>
 
'''Cytotoxicity:'''<br>
−
This Biobrick includes all enzymes of the pathway which leads to caffeine. We investigated the toxicity of caffeine and found out, that the growth of yeast strains was inhibited at concentrations of 10 mM. Caffeine with a concentration of 100 mM even showed a lethal effect on yeast cells. Lower concentrations of caffeine (range of mikro molar) did not show any growth inhibition or effect.
+
This Biobrick includes all enzymes of the pathway which leads to caffeine. We investigated the toxicity of caffeine and found out, that the growth of yeast strains was inhibited at concentrations of 10 mM. Caffeine with a concentration of 100 mM even showed a lethal effect on yeast cells. Lower concentrations of caffeine (range of micro molar) did not show any growth inhibition or effect.
 
<!--none/not known/cytotoxic for ''organism name''-->
 
<!--none/not known/cytotoxic for ''organism name''-->
  
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'''Source:'''<br>
 
'''Source:'''<br>
 +
*Preexisting BioBrick BBa_K801073,BBa_K801074, BBa_K801075
 
<!--*Commercial system: plasmid name, system name, company name-->
 
<!--*Commercial system: plasmid name, system name, company name-->
 
<!--*Plasmid: p???, provided by ?name_of_person?, ?institute/university?, ?country?-->
 
<!--*Plasmid: p???, provided by ?name_of_person?, ?institute/university?, ?country?-->
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'''Organism:'''<br>
 
'''Organism:'''<br>
 +
* Genesequence derived from ''coffea arabica''
 +
* codonoptimized for ''saccharomyces cerevisiae''
 +
*Designed for the following Chassis: ''saccharomyces cerevisiae''
 
<!--*Genesequence derived from ''?organism_name?''-->
 
<!--*Genesequence derived from ''?organism_name?''-->
 
<!--*Codonoptimized for ''?organism_name?''-->
 
<!--*Codonoptimized for ''?organism_name?''-->
 
<!--*Designed for the following Chassis: ''?organism-name?''-->
 
<!--*Designed for the following Chassis: ''?organism-name?''-->
 
<!--*Statement about functionality in other chassis.-->
 
<!--*Statement about functionality in other chassis.-->
−
 
  
 
===References===
 
===References===
Line 83: Line 101:
  
 
'''Literature references:'''<br>
 
'''Literature references:'''<br>
 +
Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol, 132(1):372–80.
 
<!--*[http://www.ncbi.nlm.nih.gov/pubmed/?PMID? '''Pubmed:''' ?Author(s)?, ?year?: ?title?]-->
 
<!--*[http://www.ncbi.nlm.nih.gov/pubmed/?PMID? '''Pubmed:''' ?Author(s)?, ?year?: ?title?]-->
  
 
'''Database references:'''<br>
 
'''Database references:'''<br>
 +
* NCBI
 
<!--*[http://www.ncbi.nlm.nih.gov/nuccore/?accessNr? '''GenBank''': ?title?]-->
 
<!--*[http://www.ncbi.nlm.nih.gov/nuccore/?accessNr? '''GenBank''': ?title?]-->
 
<!--*[http://www.ebi.ac.uk/interpro/IEntry?ac=?accessNr? '''Interpro''': ?title?]-->
 
<!--*[http://www.ebi.ac.uk/interpro/IEntry?ac=?accessNr? '''Interpro''': ?title?]-->

Latest revision as of 10:26, 24 October 2012

Caffeine Synthesis Pathway pTEF2-XMT1-tADH1-pTEF1-MXMT1-tADH1-pTEF2-DXMT1-tADH1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3613
    Illegal BglII site found at 5829
    Illegal BglII site found at 5925
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 138
    Illegal NgoMIV site found at 4317
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2385


Composite part of BBa_K801010 BBa_K801070 BBa_K801012 BBa_K319003 BBa_K801071 BBa_K801012 BBa_K801010 BBa_K801072 BBa_K801012


Keywords: caffeine, TEF1, TEF2, ADH1, expression cassette


Abbreviations:

  • CaXMT1 = coffea arabica xanthosine N-methyltransferase 1
  • CaMXMT1 = coffea arabica 7-methylxanthine N-methyltransferase 1
  • CaDXMT1 = coffea arabica 3,7-dimethylxanthine N-methyltransferase 1

Design Notes

Related BioBrick: BBa_K801073 BBa_K801074 BBa_K801075 BBa_K801076

Quality control measures:
Test digest has been performed with use of Xba1 and Pst1 (see mainpage) Part hasn´t been sequenced

Backbone:

  • Backbone name: pSB1C3'
  • Resistance: Cp

Protein coding:

  • Protein: CaXMT1-Strep-tag, CaMXTM1-Strep-tag, CaDXMT1-Strep-tag

Enzymatic activity:

  • see above related biobricks

Cytotoxicity:
This Biobrick includes all enzymes of the pathway which leads to caffeine. We investigated the toxicity of caffeine and found out, that the growth of yeast strains was inhibited at concentrations of 10 mM. Caffeine with a concentration of 100 mM even showed a lethal effect on yeast cells. Lower concentrations of caffeine (range of micro molar) did not show any growth inhibition or effect.

Safety notes:

Intellectual property:

Corresponding part author/authors:

Source

Source:

  • Preexisting BioBrick BBa_K801073,BBa_K801074, BBa_K801075


Organism:

  • Genesequence derived from coffea arabica
  • codonoptimized for saccharomyces cerevisiae
  • Designed for the following Chassis: saccharomyces cerevisiae

References

Literature references:
Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol, 132(1):372–80.

Database references:

  • NCBI