Device

Part:BBa_K3629023

Designed by: Arshia Mostoufi   Group: iGEM20_Calgary   (2020-10-13)
Revision as of 02:43, 14 October 2020 by ArshiaMostoufi (Talk | contribs) (→‎Usage and Biology)


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

2-isopropylmalate synthase (LEU4) expression construct with Gibson homology, TEF intronic promoter (BBa_K3629001)double XPR2 terminator(BBa_K3629004),Yarrowia lipolytica LEU4 coding sequence (BBa_K3629019) and reverse gPCR primer binding site for Yarrowia lipolytica.

Usage and Biology

2-isopropylmalate synthase is the first enzyme involved in the biosynthesis of leucine. This is a very important enzyme in the pathway as it serves as a regulatory point in the biosynthesis pathway. The protein has a leucine binding domain which allows for down regulation of the pathway through negative-feedback inhibition of the enzyme. Expression of this enzyme especially on a strong promoter could be used to upregulate leucine synthesis and produce a leucine overproducing strain.

Expression of LEU4 is under the TEF intronic promoter which is the natural promoter found in the wild-type Yarrowia lipolytica for the Translation Elongation Factor 1 (TEF1) gene; it is a strong constitutive promoter. It also contains the first intron of the gene for stronger expression which helps with the overexpression of LEU4. XPR2 was used in the construct which is the terminator sequence from the Yarrowia lipolytica alkaline extracellular protease XRP2 gene.

Design

Sequence and Features


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]

References

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Categories
Parameters
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