Difference between revisions of "Part:BBa K801076:Design"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K801076 short</partinfo>
 
<partinfo>BBa_K801076 short</partinfo>
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<partinfo>BBa_K801076 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K801076 SequenceAndFeatures</partinfo>
  
 +
Composite part of BBa_K801010, BBa_K801070, BBa_K801012, BBa_K319003, BBa_K801071 and BBa_K801012
 +
 +
<br>'''Keywords:'''
 +
<!--These keywords are necessary to find your part using a fulltext sarch.-->
 +
caffeine, TEF1, TEF2, ADH1, expression cassette
  
 +
<br>'''Abbreviations:'''
 +
<!--*used_abbreviation_1 = full_name_of_used_abbreviations_1-->
 +
<!--*used_abbreviation_2 = full_name_of_used_abbreviations_2-->
 +
* CaXMT1 = coffea arabica xanthosine N-methyltransferase 1
 +
* CaMXMT1 = coffea arabica 7-methylxanthine N-methyltransferase 1
 
===Design Notes===
 
===Design Notes===
−
-
 
  
 +
'''Related BioBrick:'''
 +
[https://parts.igem.org/Part:BBa_K801073 BBa_K801070]
 +
[https://parts.igem.org/Part:BBa_K801076 BBa_K801071]
 +
[https://parts.igem.org/Part:BBa_K801073 BBa_K801077]
 +
<!--*Other versions:[https://parts.igem.org/wiki/index.php?title=Part:BBa_?????? BBa_??????: Name_of_part] -->
 +
<!--*Related BioBricks:[https://parts.igem.org/wiki/index.php?title=Part:BBa_?????? BBa_??????: Name_of_part]
  
 +
'''Cloning details:'''<br>
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* Desigend in RFC 10
 +
<!--*Designed in RFC10/RFC23/RFC25/RFC25_N-part-->
 +
<!--*Mutation C889G to delete XbaI restriction site-->
 +
<!--*Truncation upstream/downstream compared to template, ?explanation?-->
 +
 +
'''Quality control measures:'''<br>
 +
* Test digest has been performed with use of Xba1 and Pst1 (see mainpage)
 +
<!--*Test digestion using ?enzyme1? & ?enzyme2?/Not yet performed-->
 +
<!--*Sequencing using primer ?primer_name?/Not yet sequenced-->
 +
<!--*Part was partly sequenced/Part was totally sequenced-->
 +
 +
'''Backbone:'''<br>
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*Backbone name: pSB1C3'
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* Resistance: Cp
 +
<!--*Resistance: Amp/Cp/Kan/Tet-->
 +
<!--*Copynumber: low/medium/high-->
 +
 +
'''Protein coding:'''<br>
 +
* Protein: CaXMT1-Strep-tag and CaMXMT1-Strep-tag
 +
 +
<!--*Protein: ?Name_of_gene_product? [Nucleotide 1 to ???]-->
 +
<!--*The protein has the amino acid replacements ???99??? to ???99???.-->
 +
<!--*The protein encoded is posttranslationally modified by ???.-->
 +
<!--*Tag: n-terminally fused/c-terminally fused His5/His6/Strep/Flag/other-->
 +
 +
'''Enzymatic activity:'''
 +
EC-number [http://enzyme.expasy.org/EC/2.1.1.158 2.1.1.158]
 +
EC-number [http://enzyme.expasy.org/EC/2.1.1.160 2.1.1.160]
 +
 +
<!--none/EC-number ?.?.?.?-->
 +
 +
'''Cytotoxicity:'''<br>
 +
This Biobrick is one part of the pathway, which leads to caffeine (the BioBrick BBa_K801075 is further needed). 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''-->
 +
 +
'''Safety notes:'''<br>
 +
<!--Known and anticipated sefety issues: none/health_risk/environmental_risk/other_risk-->
 +
<!--Known and anticipated security issues: none/other.-->
 +
 +
'''Intellectual property:'''
 +
<!--Information on patent situation.-->
 +
<!--Intellectual property claims made by the authors.-->
 +
 +
'''Corresponding part author/authors:'''
 +
<!--https://igem.org/User_Information.cgi?user_id=????/email-->
  
 
===Source===
 
===Source===
  
−
Composite part of BBa_K801010, BBa_K801070, BBa_K801012, BBa_K319003, BBa_K801071 and BBa_K801012
+
'''Source:'''<br>
 +
* Preexisting BioBrick BBa_K801070 and BBa_K801071
 +
<!--*Commercial system: plasmid name, system name, company name-->
 +
<!--*Plasmid: p???, provided by ?name_of_person?, ?institute/university?, ?country?-->
 +
<!--*Preexisting BioBrick ?Bba_number?-->
 +
<!--*cDNA Clone: ?clone_name?, ?company_name?-->
 +
<!--*Synthesized by ?company_name?.-->
 +
 
 +
<!--'''Forward Primer:'''<br><code>5'- ??? - 3'</code><br>-->
 +
<!--'''Reverse Primer:'''<br><code>5'- ??? - 3'</code><br>-->
 +
 
 +
'''Organism:'''<br>
 +
* Genesequence derived from ''Coffea arabica''
 +
* codonoptimized for ''Saccharomyces cerevisiae''
 +
* designed for ''Saccharomyces cerevisiae''
 +
 
 +
<!--*Genesequence derived from ''citrus limonen''-->
 +
<!--*Codonoptimized for ''?organism_name?''-->
 +
<!--*Designed for the following Chassis: ''?organism-name?''-->
 +
<!--*Statement about functionality in other chassis.-->
  
 
===References===
 
===References===
 +
<!-- Here you find templates to insert references to literature and different databases-->
 +
 +
'''Literature references:'''<br>
 +
*[http://www.ncbi.nlm.nih.gov/pubmed/12746542 '''Pubmed:''' 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.]
 +
*[http://www.ncbi.nlm.nih.gov/pubmed/16247553 '''Pubmed:''' Uefuji H., Ogita S., Yamaguchi Y., Koizumi N. and Sano H., 2005: Caffeine production in tobacco plants by simultaneous expression of three coffee N-methyltrasferases and its potential as a pest repellant.]
 +
*[http://www.ncbi.nlm.nih.gov/pubmed/16925551 '''Pubmed:'''  Kuranda K, Leberre V, Sokol S, Palamarczyk G, François J., 2006: Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways.]
 +
<!--*[http://www.ncbi.nlm.nih.gov/pubmed/?PMID? '''Pubmed:''' ?Author(s)?, ?year?: ?title?]-->
 +
 +
'''Database references:'''<br>
 +
* NCBI
 +
<!--*[http://www.ncbi.nlm.nih.gov/nuccore/?accessNr? '''GenBank''': ?title?]-->
 +
<!--*[http://www.ebi.ac.uk/interpro/IEntry?ac=?accessNr? '''Interpro''': ?title?]-->
 +
<!--*[http://www.uniprot.org/uniprot/?accessNr? '''Uniprot''': ?title?]-->
 +
<!--*[http://pfam.sanger.ac.uk/family/?accessNr? '''Pfam:''' ?title?]-->
 +
<!--*[http://www.rcsb.org/pdb/explore/explore.do?structureId=?accessNR? '''PDB:''' ?tile?]-->
 +
<!--*[http://www.brenda-enzymes.info/php/result_flat.php4?ecno=?accessNr? '''Branda:''' ?title?]-->

Latest revision as of 10:32, 24 October 2012

Yeast expression cassette for CaXMT1 and CaMXMT1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3613
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 138
  • 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 and 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

Design Notes

Related BioBrick: BBa_K801070 BBa_K801071 BBa_K801077

Quality control measures:

  • Test digest has been performed with use of Xba1 and Pst1 (see mainpage)

Backbone:

  • Backbone name: pSB1C3'
  • Resistance: Cp

Protein coding:

  • Protein: CaXMT1-Strep-tag and CaMXMT1-Strep-tag


Enzymatic activity: EC-number [http://enzyme.expasy.org/EC/2.1.1.158 2.1.1.158] EC-number [http://enzyme.expasy.org/EC/2.1.1.160 2.1.1.160]


Cytotoxicity:
This Biobrick is one part of the pathway, which leads to caffeine (the BioBrick BBa_K801075 is further needed). 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_K801070 and BBa_K801071


Organism:

  • Genesequence derived from Coffea arabica
  • codonoptimized for Saccharomyces cerevisiae
  • designed for Saccharomyces cerevisiae


References

Literature references:

  • [http://www.ncbi.nlm.nih.gov/pubmed/12746542 Pubmed: 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.]
  • [http://www.ncbi.nlm.nih.gov/pubmed/16247553 Pubmed: Uefuji H., Ogita S., Yamaguchi Y., Koizumi N. and Sano H., 2005: Caffeine production in tobacco plants by simultaneous expression of three coffee N-methyltrasferases and its potential as a pest repellant.]
  • [http://www.ncbi.nlm.nih.gov/pubmed/16925551 Pubmed: Kuranda K, Leberre V, Sokol S, Palamarczyk G, François J., 2006: Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways.]

Database references:

  • NCBI