Regulatory

Part:BBa_K3629002

Designed by: Sravya Kakumanu   Group: iGEM20_Calgary   (2020-10-11)
Revision as of 22:39, 27 October 2020 by Sravyakakumanu (Talk | contribs) (→‎References)

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Yarrowia lipolytica EXP Promoter

Promoter from the Yarrowia lipolytica EXP gene (an extracellular protease).

Usage and Biology

EXP promoter is a native Yarrowia lipolytica promoter for the EXP gene. Is one of the strongest constitutive promoters found in the organism. EXP promoter is often used for the expression of heterologous proteins in Y. lipolytica due to its high transcriptional level (1).

This promoter was used in the TrEGII expression construct (BBa_K3629017) as this promoter, in combination with the XPR2 signal peptide (BBa_K3629000), has been shown to secrete high levels of TrEGII in Y. lipolytica (up to 132mg/L) (2). This promoter is thought not to be as strong as TEFin (BBa_K3619001) promoter (2) but has approximately 1.2x greater activity than the wild-type TEF1 (BBa_K211700) promoter (2).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 1025
    Illegal SpeI site found at 602
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NheI site found at 663
    Illegal SpeI site found at 602
    Illegal SpeI site found at 1026
    Illegal PstI site found at 1040
    Illegal NotI site found at 7
    Illegal NotI site found at 1033
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 1026
    Illegal SpeI site found at 602
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 602
    Illegal SpeI site found at 1026
    Illegal PstI site found at 1040
  • 1000
    COMPATIBLE WITH RFC[1000]

There is a SpeI site within this promoter sequence, making it RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (EcoRI, NotI, XbaI, and PstI) can be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly.

References

1. 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

2. Wei H, Wang W, Alper HS, et al. Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone. Front Microbiol. 2019;9:3276. Published 2019 Jan 9. doi:10.3389/fmicb.2018.03276


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