Part:BBa_K4452025
Ferritin with prSSG1 transit peptide + RUBY + nptII
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.
GoldenBraid is a standardized assembly system based on type IIS restriction enzymes “that allows the indefinite growth of composite parts through the succession of iterative assembling steps.” This criteria is important for us because our cloning plan requires assembling basic parts into three distinct genes and then assembling those genes together. Additionally, GoldenBraid was designed with the intention of being an assembly standard for plant synthetic biology [17], so it would be an appropriate method for our project.
Implementation of GoldenBraid requires (1) specific type IIS restriction sites on basic parts and (2) specific destination plasmids with type IIS restriction sites positioned inside the vectors to allow for “braiding” parts binarily in indefinite successive iterations.
Basic parts are flanked with BsaI recognition-cleavage sites using distinct 4 bp cleavage sequences for neighboring basic parts such that the parts can be assembled in a specified sequence. When the parts are ligated with the correct destination plasmid that is flanked by BsaI sites in divergent orientation, all BsaI recognition sites disappear from the resulting expression plasmid. This process of putting the parts into a destination plasmid with BsaI digestion is referred to as level alpha assembly.
To assemble parts from level alpha plasmids into another destination plasmid requires BsmBI digestion, this is referred to as level omega assembly. The level alpha plasmids and the level omega destination plasmid will be flanked by complementary 4 bp BsmBI cleavage sites.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3712
Illegal NgoMIV site found at 4291
Illegal NgoMIV site found at 6004 - 1000INCOMPATIBLE 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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