Journal: Plant Physiology

Article Title: MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology ^{1 [OPEN]}

doi: 10.1104/pp.18.01220

Figure Lengend Snippet: Schematic representation of Golden Gate assembly using the MoChlo kit. A, Four vectors containing different Level-0 modules flanked by BsaI sites. P, Promoter (blue); U, 5ʹUTR (gray); GOI, gene of interest (red); T, terminator/3ʹUTR (black). B, Level-1 Golden Gate assembly. The BsaI restriction digestion releases Level-0 parts (P1–P7, U1–U7, GOI1–GOI7, and T1–T7) equipped with 5ʹ and 3ʹ overhangs (I–V) used for directional cloning. The 5ʹ and 3ʹ overhangs (I and V, respectively) of P-U-GOI-T cassettes (C1–C7) are compatible with Level-1 vectors (V1–V7). The Level-1 assembly results in seven Level-1 vectors containing seven cassettes (C1–C7) flanked by BpiI sites. C, Level-2 Golden Gate assembly. The BpiI restriction digestion of Level-1 vectors releases cassettes (C1–C7) equipped with 5ʹ and 3ʹ overhangs (1–8) able to provide directional cloning from C1 to C7. An end linker (L7; green) equipped with compatible 5ʹ and 3ʹ overhangs (8 and 9, respectively) allows integration of the Level-2 operon in between the homologous sequences (H1 and H2) of a chloroplast transformation vector (Chl-Vector; destination vector Level-2).

Article Snippet: All sequences were domesticated for use in the MoChlo kit as previously described, and the sequences of all Level-0 modules are included in Supplemental Table S1 , with the entire MoChlo kit available from Addgene.

Techniques: Clone Assay, Transformation Assay, Plasmid Preparation

Journal: Plant Physiology

Article Title: MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology ^{1 [OPEN]}

doi: 10.1104/pp.18.01220

Figure Lengend Snippet: Level-0 parts provided in the MoChlo kit A detailed description of all Level-0 parts, including nucleotide sequences, is included in Supplemental Table S1 . (–), No data; u, unpublished; NEP, nuclear encoded polymerase; PEP, plastid encoded polymerase; DB, downstream box; Ec, Escherichia coli; TYMV, turnip yellow mosaic virus; BMV, brome mosaic virus; AMV, Alfalfa mosaic virus; TMV, tobacco mosaic virus; SL, stem-loop; codon opt., plastid codon optimized.

Article Snippet: All sequences were domesticated for use in the MoChlo kit as previously described, and the sequences of all Level-0 modules are included in Supplemental Table S1 , with the entire MoChlo kit available from Addgene.

Techniques: Functional Assay

Journal: Plant Physiology

Article Title: MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology ^{1 [OPEN]}

doi: 10.1104/pp.18.01220

Figure Lengend Snippet: Schematic of the MoChlo kit for combinatorial assembly of operons for metabolic engineering. In the current iteration of the kit, combinatorial assembly of individual cassettes (Level-1 constructs) can be conducted using 47 promoters, 38 5ʹUTRs, nine promoter:5ʹUTR fusions, and 10 terminators/3ʹUTRs. In addition, the user has the ability to assemble up to seven individual cassettes, each with its own regulatory elements and GOIs. Furthermore, during Level-2 assembly, the user can choose from three different integration sites (trnI-trnA, trnV-3ʹrps12, and trnG-trnfM) from three different plant species (tobacco, maize, and potato). After completing assembly, the user would then bombard the Level-2 constructs into leaf discs and select and regenerate transplastomic plants.

Article Snippet: All sequences were domesticated for use in the MoChlo kit as previously described, and the sequences of all Level-0 modules are included in Supplemental Table S1 , with the entire MoChlo kit available from Addgene.

Techniques: Construct