Device

Part:BBa_K3629023:Design

Designed by: Arshia Mostoufi   Group: iGEM20_Calgary   (2020-10-13)


2-isopropylmalate synthase (LEU4) overexpression construct with Gibson homology


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 339
    Illegal EcoRI site found at 2875
    Illegal SpeI site found at 569
    Illegal PstI site found at 534
    Illegal PstI site found at 986
    Illegal PstI site found at 2144
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 339
    Illegal EcoRI site found at 2875
    Illegal NheI site found at 91
    Illegal SpeI site found at 569
    Illegal PstI site found at 534
    Illegal PstI site found at 986
    Illegal PstI site found at 2144
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 339
    Illegal EcoRI site found at 2875
    Illegal BglII site found at 1629
    Illegal XhoI site found at 1329
    Illegal XhoI site found at 1878
    Illegal XhoI site found at 2136
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 339
    Illegal EcoRI site found at 2875
    Illegal SpeI site found at 569
    Illegal PstI site found at 534
    Illegal PstI site found at 986
    Illegal PstI site found at 2144
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 339
    Illegal EcoRI site found at 2875
    Illegal SpeI site found at 569
    Illegal PstI site found at 534
    Illegal PstI site found at 986
    Illegal PstI site found at 2144
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The construct is flanked with two Gibson homology sequences. Upon digestion with BbsI, the construct can be connected to the nourseothricin resistance expression construct (BBa_K3629015) on the 5' side and to either the mCherry (BBa_K3629025) or mCitrine (BBa_K3629026) expression construct on the 3' side. This allows for both a selection marker and a fluorescence protein to be easily added to the construct in one Gibson reaction.

The construct was designed to be ultimately integrated into the genome of Yarrowia lipolytica. Therefore, a pair of gPCR primers can be used to help determine the location of the integration of the construct in the genome. The forward gPCR primer binding site is found in the nourseothricin resistance expression construct (BBa_K3629015) while the reverse gPCR primer binding site is located at the end of this construct (LEU4 Overexpression construct).

An extra XPR2 terminator (BBa_K3629004) was included at the beginning of the construct, right before the promoter in case the construct is integrated into the coding sequence of a gene in the genome. This would stop the expression of that gene which might have otherwise interfered with the expression of the LEU4 gene.

LEU4 coding sequence was also codon-optimized for expression and function in Yarrowia lipolytica.

Source

The TEF intronic promoter (BBa_K3629001)double XPR2 terminator(BBa_K3629004), and LEU4 coding sequence (BBa_K3629019) were all obtained from genome sequence of Yarrowia lipolytica CDS: T01033.

References

1.[online] https://www.genome.jp/kegg-bin/show_pathway?yli00400 (Accessed October 26, 2020)

2.Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11

3.Czajka, Jeffrey J, Nathenson, Justin A, Benites, Veronica T, Baidoo, Edward E. K, Cheng, Qianshun, Wang, Yechun, & Tang, Yinjie J. (2018). Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone. Microbial Cell Factories, 17(1), 136–136. https://doi.org/10.1186/s12934-018-0984-x