Generator

Part:BBa_K801076:Design

Designed by: Dennis Hell   Group: iGEM12_TU_Munich   (2012-09-21)
Revision as of 10:27, 24 October 2012 by Dennis48 (Talk | contribs)

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, 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