Part:BBa_K801073:Design

CaXMT1 expression cassette for yeast

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 138
  
• 1000
  COMPATIBLE WITH RFC[1000]

Composite part of BBa_K801010, BBa_K801070, BBa_K801012

Keywords: CaXMT1, TEF2, ADH1, caffeine,

Abbreviations:

• CaXMT1 = xanthosine methyltransferase
• CaXMT1 = coffea arabica xanthosine N-methyltransferase 1

Design Notes

Related BioBrick:

• Other versions:
  • BBa_K801070: xanthosine methyltransferase CaXMT1-strep
• Related BioBricks:
  • BBa_K801071: 7-methylxanthine N-methyltransferase CaMXMT1-strep
  • BBa_K801072: 3,7-dimethylxanthine N-methyltransferase CaDXMT1-strep
  • BBa_K801073: CaXMT1 expression cassette for yeast
  • BBa_K801074: CaMXMT1 expression cassette for yeast
  • BBa_K801075: CaDXMT1 expression cassette for yeast
  • BBa_K801076: Yeast expression cassette for CaXMT1 and CaMXMT1
  • BBa_K801077: Caffeine Synthesis Pathway pTEF2-XMT1-tADH1-pTEF1-MXMT1-tADH1-pTEF2-DXMT1-tADH1

Cloning details:

• Designed in RFC10

Quality control measures:

• Test digestion using XbaI & PstI
• Part was totally sequenced

Backbone:

• Backbone name: pSB1C3
• Resistance: Cp
• Copynumber: high

Protein coding:

• Protein: CaXMT1 [Nucleotide 663 to 1778]
• Tag: c-terminally fused Strep

Enzymatic activity:

• EC-number: 2.1.1.158

Cytotoxicity:
This Biobrick is one part of the pathway, which leads to caffeine (the BioBricks BBa_K801074 and BBa_K801075 are 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

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