Composite

Part:BBa_K4452018:Design

Designed by: iGEM22_Hopkins   Group: iGEM22_Hopkins   (2022-09-30)
Revision as of 04:17, 10 October 2022 by KalenClifton (Talk | contribs) (→‎Design Notes)

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nptII antibiotic resistance for expression in plants


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 226
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1472
    Illegal BsaI.rc site found at 1751


Design Notes

This composite part was assembled using Golden Braid assembly, specifically level alpha assembly with BsaI.

Part Description BsaI site
BBa_J428074 Plant promoter CaMV35S GGAG / TACT
BBa_J428088 Plant fiveUTRs AtRbcS2B 5UTR TACT / AATG
BBa_J428077 nptII AATG / GCTT
BBa_J428082 Plant threeUTRs Tnos GCTT / CGCT
BBa_J428343 pJUMP29-1C GGAG / TACT

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 [1], 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.

Source

none

References

[1] Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, et al. (2011) GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules. PLOS ONE 6(7): e21622. https://doi.org/10.1371/journal.pone.0021622