Composite

Part:BBa_M50434:Design

Designed by: Sainiteesh Maddineni and Mohammed Osman   Group: Stanford BIOE44 - S11   (2018-04-25)


Oxalate Catabolism Actuator in S. cerevisiae via AtAAE3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 835
    Illegal SpeI site found at 1075
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 835
    Illegal SpeI site found at 1075
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 835
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 835
    Illegal SpeI site found at 1075
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 835
    Illegal SpeI site found at 1075
    Illegal AgeI site found at 150
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Our goal was to have production of AtAAE3 under tight inductive control, so a galactose-inducible, glucose-repressible pGAL1 promoter was utilized. An ATUM-default yeast Kozak consensus sequence (aAaAaAATGTCt) was utilized with minor modifications. Specifically, we deleted the last 5 nucleotides since our AtAAE3 gene already contains a start codon. Note that we incorporated the Kozak into our gene itself (Part: BBa_M50433). The pGAL1 promoter was also We also used ATUM’s default pGAL1 promoter (iGEM Part: BBa_J63006). Our coding sequence also had a 6X HIS tag inserted at the C-terminus. Moreover, the terminator utilized was also a default ATUM terminator for S. cerevisiae (iGEM Part: K1462070). The plasmid vector from ATUM utilized was pD1201. Overall, our basic parts were optimized for yeast, since previous literature conducted similar oxalate experiments in yeast. An E. coli glycerol stock (Barcode: 0133024479) for culture in LB + kanamycin media and a yeast glycerol stock for culture in 2% raffinose (Barcode: 0133011812) were made by mixing cultures in 1:1 ratio with 50% glycerol.

Source

The coding sequence comes from the analogous oxalyl-CoA synthetase of the species Arabidopsis thaliana. The sequence of for the AtAAE3 gene (iGEM Part: BBa_M50433) was derived from the European Nucleotide Archive via UniProtKB (UniProtKB Entry: Q9SMT7). The actual sequence was submitted by Salanoubat et al. We then codon-optimized this sequence for S. cerevisiae, the organism we engineered, and added a 6X HIS tag to the end of the coding sequence for Western Blotting ourselves.

pGAL1 promoter: iGEM Part: BBa_J63006

Terminator: iGEM Part: K1462070

References

http://parts.igem.org/Part:BBa_J63006

http://parts.igem.org/Part:BBa_K1462070

http://parts.igem.org/Part:BBa_M50433

Foster J, Nakata PA 2014. An oxalyl-CoA synthetase is important for oxalate metabolism in Saccharomyces cerevisiae. FEBS Lett. 588(1):160-6.

Salanoubat M, et al. 2000. Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana. Nature. 408(6814):820-2.

Inoue K, Aikawa S, Fukushima Y (2018). Colorimetric detection of oxalate in aqueous solution by a pyrogallol red-based Cu(2+) complex. Luminescence. 33(2):277-281.

Blobel F, Erdmann R 1996. Identification of a yeast peroxisomal member of the family of AMP-binding proteins. Eur J Biochem. 240(2):468-76.

Paulo JA, O'Connell JD, Gaun A, Gygi SP 2015. Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae. Mol Biol Cell. 26(22):4063-74.