Part:BBa_K4452021:Design
Ferritin with prFB transit peptide + RUBY reporter + nptII antibiotic resistance
- 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 3095
Illegal BglII site found at 7103
Illegal BglII site found at 8187
Illegal BamHI site found at 5752 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3688
Illegal NgoMIV site found at 4267
Illegal NgoMIV site found at 5980 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2406
Illegal BsaI site found at 7498
Illegal BsaI site found at 9433
Illegal BsaI.rc site found at 2685
Illegal BsaI.rc site found at 7777
Illegal BsaI.rc site found at 9712
Design Notes
The three genes (BBa_K4452011, BBa_K4452017, BBa_K4452018) and a synthetic spacer (BBa_K4452009) were assembled into BBa_K4452021 using Golden Braid assembly, specifically level omega assembly with BsmBI.
Part | Description | BsmBI site |
---|---|---|
BBa_K4452011 | Ferritin | GGAG / AATG |
BBa_K4452017 | RUBY reporter | AATG / AGCC |
BBa_K4452018 | nptII Antibiotic Resistance | AGCC / TTCG |
BBa_K4452009 | M13R primer | TTCG / CGCT |
BBa_K4452010 | pLX-B3(omega)1 | GGAG / CGCT |
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