MoChlo: A Versatile, Modular Cloning Toolbox for Chloroplast Biotechnology

Abstract

Plant synthetic biology is a rapidly evolving field with new tools constantly emerging to drive innovation. Of particular interest is the application of synthetic biology to chloroplast biotechnology to generate plants capable of producing new metabolites, vaccines, biofuels, and high-value chemicals. Progress made in the assembly of large DNA molecules, composing multiple transcriptional units, has significantly aided in the ability to rapidly construct novel vectors for genetic engineering. In particular, Golden Gate assembly has provided a facile molecular tool for standardized assembly of synthetic genetic elements into larger DNA constructs. In this work, a complete modular chloroplast cloning system, MoChlo, was developed and validated for fast and flexible chloroplast engineering in plants. A library of 128 standardized chloroplast-specific parts (47 promoters, 38 5' untranslated regions [5'UTRs], nine promoter:5'UTR fusions, 10 3'UTRs, 14 genes of interest, and 10 chloroplast-specific destination vectors) were mined from the literature and modified for use in MoChlo assembly, along with chloroplast-specific destination vectors. The strategy was validated by assembling synthetic operons of various sizes and determining the efficiency of assembly. This method was successfully used to generate chloroplast transformation vectors containing up to seven transcriptional units in a single vector (∼10.6-kb synthetic operon). To enable researchers with limited resources to engage in chloroplast biotechnology, and to accelerate progress in the field, the entire kit, as described, is available through Addgene at minimal cost. Thus, the MoChlo kit represents a valuable tool for fast and flexible design of heterologous metabolic pathways for plastid metabolic engineering.

Figure 1.

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).

Figure 2.

Level-1 cloning for a four-gene operon-1. A, Schematic representation of a Level-1 cassette: promoter (P; blue); 5ʹUTR (5ʹ; gray); GOIs (GOI1–GOI3 or aadA; red); terminator/3ʹUTR (3ʹ; black). BpiI sites flanking the Level-1 cassette are indicated. B to E, PCR to confirm Level-1 assembly. Specific primers for the internal gene (B, GOI-1; C, GOI-2; D, GOI-3; E, aadA) and the promoter/3ʹUTR of cassettes 1A, 1B, 1C, and 1D (B, C. 1A; C, C. 1B; D, C. 1C; E, C. 1D) have been used. DNA bands at the predicted molecular mass (kb) confirm correct Level-1 assembly of all cassettes. Negative controls (blanks) of PCR images B to E are indicated. F, BpiI restriction digestion of Level-1 vectors. The four Level-1 cassettes released after digestion are indicated with asterisks (size indicated in Table 1). The empty vector 1 (vector position 1 forward) is used as a positive control. The LacZα fragment (0.616 kb) release after digestion of vector 1 is indicated with the triangle. The molecular mass (kb) of the backbone vector is indicated with black arrows. DNA markers are shown in each image.

Figure 3.

Level-2 cloning for operon-1. A, Schematic representation of Level-2 operon-1: promoter (P; blue); 5ʹUTR (5ʹ; gray); GOIs (GOI1–GOI3; red); gene for selection (aadA; yellow); terminator/3ʹUTR (3ʹ; black). Level-1 cassettes 1A, 1B, 1C, and 1D are assembled in positions 1, 2, 3, and 4 of operon-1, respectively. The operon-1 is cloned in a Level-2 vector between left (L; 1.17 kb) and right (R; 1.45 kb) arms homologous to the trnG/trnfM site of the potato plastome. B to E, PCR to confirm the presence of all genes of operon-1 (three purified vectors; 1–3). Specific primers for GOI-1 (B), GOI-2 (C), GOI-3 (D), and aadA (E) have been used. DNA bands at the predicted molecular mass (kb; black arrows) in both purified plasmid (operon-1) and positive controls (positive C.) confirm correct Level-2 assembly of operon-1. F to J, PCR to confirm the correct order of four genes in the operon-1. Pairs or primers specific for trnG/GOI-1 (F), GOI-1/GOI-2 (G), GOI-2/GOI-3 (H), GOI-3/aadA (I), and aadA/trnfM (J) have been used. DNA bands at the predicted molecular mass confirm the correct order (kb; black arrows). Negative controls (blanks) for PCR and DNA markers are shown in B to J.

Figure 4.

Level-1 cloning for the Lux operon for plastid engineering. A, Schematic representation of a Level-1 cassette: promoter (P; blue); 5ʹUTR (5ʹ; gray); Lux (C, D, A, B, E, or G) or aadA gene for selection (red); terminator/3ʹUTR (3ʹ; black). BpiI sites flanking the Level-1 cassette are indicated. B to H, PCR to confirm Level-1 assembly. Specific primers for Lux genes (B, Lux-C; C, Lux-D; D, Lux-A; E, Lux-B; F, Lux-E; G, Lux-G), the gene for selection (H; aadA), and the promoter/3ʹUTR of cassettes LC1, LD1, LA1, LB1, LE1, LG1, and S1 (B, C. LC1; C, C. LD1; D, C. LA1; E, C. LB1; F, C. LE1; G, C. LG1; H, C. S1) have been used. DNA bands at the predicted molecular mass (kb; black arrows) confirm correct Level-1 assembly of all cassettes. Negative controls (blanks) of PCR images B to H are indicated. I, BpiI restriction digestion of Level-1 vectors. Level-1 cassettes released after digestion are indicated with asterisks (cassette size indicated in Table 2). Empty vector 1 (position 1 forward) was used as a positive control. The LacZα fragment release after digestion of vector 1 is indicated with the arrowhead (0.616 kb). The molecular mass (kb) of the backbone vector is indicated with black arrows. DNA markers are shown in B to I.

Figure 5.

Level-2 cloning for the seven-gene Lux operon. A, Schematic representation of Level-2 assembly of Lux operon-1: promoter (P; blue); 5ʹUTR (5ʹ; gray); Lux (C, D, A, B, E, or G; red); gene for selection (aadA; yellow); terminator/3ʹUTR (3ʹ; black). Level-1 cassettes LC1, LD1, LA1, LB1, LE1, LG1, and S1 are assembled in operon positions 1, 2, 3, 4, 5, 6, and 7, respectively. The Lux operon-1 is cloned in Level-2 vector between left (L; 1.17 kb) and right (R; 1.45 kb) arms homologous to the trnG/trnfM site of the potato plastome. B to H, PCR of all genes of the Lux operon-1. Primers specific for Lux-C (B), Lux-D (C), Lux-A (D), Lux-B (E), Lux-E (F), Lux-G (G), and aadA (H) have been used. DNA bands at the predicted molecular mass (kb; black arrows) for both purified plasmid (Lux operon-1) and positive controls (positive C.) confirmed the presence of all genes. I to P, PCR to check correct gene order in the Lux operon-1. Pairs or primers specific for trnG/Lux-C (I), Lux-C/Lux-D (J), Lux-D/Lux-A (K), Lux-A/Lux-B (L), Lux-B/Lux-E (M), Lux-E/Lux-G (N), Lux-G/aadA (O), and aadA/trnfM (P) have been used. DNA bands (black arrows) at the predicted molecular mass (kb) confirm correct gene order. Negative controls (blanks) of PCR and DNA markers are shown in B to P.

Figure 6.

An example of in vivo validation of two MoChlo-enabled constructs in potato chloroplasts. A, Schematic representation of test operons controlled by two examples of promoter::5ʹUTR combinations. The operon (red) is controlled by the rrn promoter (P1) along with a synthetic RBS as 5ʹUTR (5ʹ1; blue and gray; Operon X) or the promoter::5ʹUTR fusion of accD gene (P2:5ʹ2; blue and gray; Operon Y). The expression of mEmerald (mEme; green) is under the control of the operon promoter, while the selection gene, aadA (yellow), is under the control of both promoter::5ʹUTR (P-5ʹ[3]; blue and gray) and terminator/3ʹUTR (T; black) of the psbA gene. B to I, Confocal images of transplastomic green callus transformed with Operon X (B–E) or Operon Y (F–I). In both cases, the mEmerald fluorescent signal is localized to chloroplasts. B and F show mEmerald signal; C and G show chlorophyll autofluorescence; D and H show bright-field images; and E and I show merged images. Bars = 20 µm for all confocal images.

Figure 7.

Operon integration in the potato plastome. A, Schematic representation of chloroplast constructs: rrn promoter (P1) along with a synthetic RBS used as 5′UTR (5′1; blue and gray); promoter::5′UTR fusion of accD gene (P2:5′2; blue and gray); P-5′[3], promoter::5′UTR of the psbA gene (blue and gray); T, terminator/3′UTR of psbA gene (black); operon X and Y (red); mEme (mEmerald fluorescent marker; green); and aadA (3′-adenylyltransferase selection marker; yellow). The left (L; 1.17 kb) and right (R; 1.45 kb) arms homologous to the trnG/trnfM site of the potato plastome are indicated. B to E, PCR on total genomic DNA extracted from transplastomic green callus transformed with Operon X (B and C) or Operon Y (D and E). A pair of primers specific for the aadA gene (B and D) and aadA/trnfM (C and E) have been used to check correct operon integration. DNA bands at the predicted molecular mass confirm correct integration of both operons (kb; black arrows in B–E and black line in A). Positive and negative controls (blanks) along with potato wild-type samples and DNA markers are shown.

Figure 8.

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.