Composite

Part:BBa_K4452022

Designed by: iGEM22_Hopkins   Group: iGEM22_Hopkins   (2022-09-30)


Ferritin with prcpHsc70-1 transit peptide + RUBY + nptII

This construct is designed for implementing root magnetotropism by overexpressing ferritin in statoliths of columella cells in Arabidopsis thaliana . While no statolith import sequences have been validated, this construct includes a prcpHsc70-1 transit peptide as a candidate transit peptide sequence for importing ferritin into statoliths.

Background

To restore directional root growth in microgravity, Hopkins iGEM 2022 proposed that the existing gravitropic mechanisms can be engineered to respond to an artificial cue. We set out to engineer roots to grow in the direction of magnetic field gradients: magnetotropism.

Plants sense gravity via statoliths—starch-laden organelles in root tip columella cells—which sediment due to their weight. Statolith sedimentation triggers changes in the efflux of auxin, a universal plant hormone that induces plant cell elongation. Polarized auxin accumulation along the upper and lower sides of roots causes differential elongation of cells, guiding root growth in the direction of gravity.

We predicted that filling statoliths with iron-loading proteins, like ferritin, would allow the statoliths to move in response to a magnetic gradient. For our project we designed a genetic construct that allows for ferritin to be expressed in Arabidopsis and imported into statoliths.

Design

This specific construct is composed of three genes:

  • BBa_K4452012: Ferritin with prcpHsc70-1 transit peptide
    • Pyrococcus furiosus ferritin (PFt), an efficient iron storage protein, linked to GFP with expression under a columella cell-specific promoter, PLT-2, and with an N-terminus signal peptide to imports the ferritin into statoliths.
  • BBa_K4452017: RUBY reporter under 35S promoter
    • For visual selection of positively transformed seeds, RUBY reporter is expressed with the constitutive CMV35S plant specific promoter, a 5’ UTR from the AtRbcS2B gene, and a 3’ UTR plant terminator.
  • BBa_K4452018: nptII antibiotic resistance for expression in plants
    • For selection of positively transformed seedling on agar plates, nptII confers resistance to neomycin/kanamycin. This antibiotic resistance gene is expressed with the constitutive CMV35S plant specific promoter, a 5’ UTR from the AtRbcS2B gene, and a 3’UTR plant terminator.

Assembly

These three genes and a synthetic spacer (BBa_K4452009) were assembled into BBa_K4452022 using Golden Braid assembly, specifically level omega assembly with BsmBI.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 95
    Illegal BglII site found at 527
    Illegal BglII site found at 3059
    Illegal BglII site found at 7067
    Illegal BglII site found at 8151
    Illegal BamHI site found at 5716
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3652
    Illegal NgoMIV site found at 4231
    Illegal NgoMIV site found at 5944
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2370
    Illegal BsaI site found at 7462
    Illegal BsaI site found at 9397
    Illegal BsaI.rc site found at 2649
    Illegal BsaI.rc site found at 7741
    Illegal BsaI.rc site found at 9676


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