Composite

Part:BBa_K2909013

Designed by: Laurent CHEN   Group: iGEM19_Sorbonne_U_Paris   (2019-08-28)

HiBiT-DGAT-1-2_HiBiT-LPAAT-A_HygroR E. guineensis

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 5
    Illegal EcoRI site found at 3104
    Illegal EcoRI site found at 5618
    Illegal PstI site found at 2389
    Illegal PstI site found at 4372
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5
    Illegal EcoRI site found at 3104
    Illegal EcoRI site found at 5618
    Illegal NheI site found at 5882
    Illegal PstI site found at 2389
    Illegal PstI site found at 4372
    Illegal NotI site found at 217
    Illegal NotI site found at 3316
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5
    Illegal EcoRI site found at 3104
    Illegal EcoRI site found at 5618
    Illegal BglII site found at 2297
    Illegal BamHI site found at 2537
    Illegal BamHI site found at 4214
    Illegal BamHI site found at 5051
    Illegal XhoI site found at 758
    Illegal XhoI site found at 1020
    Illegal XhoI site found at 3857
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 5
    Illegal EcoRI site found at 3104
    Illegal EcoRI site found at 5618
    Illegal PstI site found at 2389
    Illegal PstI site found at 4372
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 5
    Illegal EcoRI site found at 3104
    Illegal EcoRI site found at 5618
    Illegal PstI site found at 2389
    Illegal PstI site found at 4372
    Illegal NgoMIV site found at 4927
    Illegal NgoMIV site found at 6583
    Illegal NgoMIV site found at 6765
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2575
    Illegal SapI.rc site found at 5089


Introduction

1- Biological background

This part is a composed of two CDS coding for the E. guineensis DGAT-1-2 (BBa_K2909003) and LPAAT-A (BBa_K2909002) enzymes, both tagged with a N-terminal HiBiT tag (BBa_K2909000).
Both enzymes are under the control of a nitrate-inducible Chlamydomonas reinhardtii promoter called NIT1.
This part also contains a Hygromycin resistance cassette called AphVII to select the recombinant clones.

This part has been designed to enhance the overall production of triglycerides in Chlamydomonas reinhardtii. The N-terminal HiBiT tag allows a quick determination of the enzyme expression.

This part is a linear DNA that is the result of the digestion of the level M plasmid by the type IIS BsaI restriction enzyme.
It is this linear DNA that is integrated into the genomic DNA of Chlamydomonas reinhardtii during the transformation.

2- Usage in iGEM projects

Bio(oil)gical Factory (iGEM Sorbonne Université 2019)

Characterization

References

  1. Schwinn, M. K. et al. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide. ACS Chem. Biol. 13, 467–474 (2018).
  2. Crozet, P. et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth. Biol. 7, 2074–2086 (2018).
  3. 1.Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6, e16765 (2011).
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Parameters
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