Composite

Part:BBa_K4452025

Designed by: iGEM22_Hopkins   Group: iGEM22_Hopkins   (2022-09-30)
Revision as of 03:44, 10 October 2022 by KalenClifton (Talk | contribs) (→‎Assembly)


Ferritin with prSSG1 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 prSSG1 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_K4452015: Ferritin with prSSG1 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_K4452025 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 3119
    Illegal BglII site found at 7127
    Illegal BglII site found at 8211
    Illegal BamHI site found at 5776
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3712
    Illegal NgoMIV site found at 4291
    Illegal NgoMIV site found at 6004
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2430
    Illegal BsaI site found at 7522
    Illegal BsaI site found at 9457
    Illegal BsaI.rc site found at 2709
    Illegal BsaI.rc site found at 7801
    Illegal BsaI.rc site found at 9736


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