CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax

Abstract

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

Figure 1.

Adaptation of the Plant Golden Gate MoClo level 0 syntax for generating level 1 assemblies for transfer to level T. A, The format for a level 0 MoClo acceptor vector with the part bordered by two BsaI sites. B, Typical level 0 parts from the Plant MoClo kit (Engler et al., 2014), where parts of the same type are bordered by the same pair of fusion sites (for each fusion site, only the sequence of the top strand is shown). Note that the parts are not drawn to scale. C and D, The syntax of the Plant MoClo kit was adapted to generate level 0 parts for engineering marked and unmarked cyanobacterial mutant strains (Lea-Smith et al., 2016). E to I, To generate knock-in mutants, short linker parts (30 bp) were constructed to allow assembly of individual flanking sequences, or marker cassettes (Ab^R or sacB), in level 1 vectors for subsequent assembly in level T. J and K, Parts required for generating synthetic srRNA or CRISPRi level 1 constructs. See Supplemental Information S2 and S3 for workflows. 3U+Ter, 3′UTR and terminator; Ab^R, antibiotic resistance cassette; Ab^R DOWN LINKER, short sequence (∼30 bp) to provide CGCT overhang; Ab^R UP LINKER, short sequence (∼30 bp) to provide GAGG overhang; CDS2(stop), coding sequence with a stop codon; DOWN FLANK, flanking sequence downstream of target site; DOWN FLANK LINKER, short sequence (∼30 bp) to provide GGAG overhang; Prom+5U, promoter and 5′ UTR; Prom TSS, promoter transcription start site; sacB, levansucrase expression cassette; sacB UP LINKER, short sequence (∼30 bp) to provide GAGG overhang; sgRNA, single guide RNA; SP, signal peptide; srRNA, small regulatory RNA; UP FLANK, flanking sequence upstream of target site; UP FLANK LINKER, short sequence (∼30 bp) to provide CGCT overhang; UNMARK LINKER, short sequence to bridge UP FLANK and DOWN FLANK.

Figure 2.

Extension of the Plant Golden Gate MoClo Assembly Standard for cyanobacterial transformation. Assembly relies on one of two Type IIS restriction endonuclease enzymes (BsaI or BpiI). Domesticated level 0 parts are assembled into level 1 vectors. Up to seven level 1 modules can be assembled directly into a level T cyanobacterial transformation vector, which consists of two subtypes (either a replicative or an integrative vector). Alternatively, larger vectors with more modules can be built by assembling level 1 modules into level M, then cycling assembly between level M and level P, and finally transferring from level P to level T. Antibiotic selection markers are shown for each level. Level T vectors are supplied with internal antibiotic selection markers (shown), but additional selection markers could be included from level 1 modules as required. See Supplemental Table S1 and Supplemental Information S4 for the full list and maps of level T acceptor vectors.

Figure 3.

Generating knock-out mutants in cyanobacteria. A, Assembled level T vector cpcBA-M (see Fig. 1C) targeting the cpcBA promoter and operon (3,563 bp) to generate a marked ΔcpcBA “Olive” mutant in Synechocystis sp. PCC 6803. Following transformation and segregation on kanamycin (∼3 months), a segregated marked mutant was isolated (wild-type [WT] band is 3,925 bp, marked mutant band is 5,503 bp, 1-kb DNA ladder [New England Biolabs] is shown). B, Assembled level T vector cpcBA-UM (see Fig. 1D) for generating an unmarked ΔcpcBA mutant. Following transformation and segregation on Suc (∼2 weeks), an unmarked mutant was isolated (unmarked band is 425 bp). C, Liquid cultures of wild type and marked and unmarked Olive mutants. D, Spectrum showing the absorbance of the unmarked Olive mutant and wild-type cultures after 72 h of growth. Values are the average of four biological replicates ± se and are standardized to 750 nm.

Figure 4.

Generating knock-in mutants in cyanobacteria. A, Assembly of level 1 modules cpcBA-UF (see Fig. 1E) in the level 1, position 1 acceptor (L1P1), P_cpc560-eYFP-T_rrnB (see Fig. 1G) in L1P2, and cpcBA-DF (see Fig. 1F) in L1P3. B, Transfer of level 1 assemblies to level T vector cpcBA-eYFP for generating an unmarked ΔcpcBA mutant carrying an eYFP expression cassette. Following transformation and segregation on Suc (∼3 weeks), an unmarked eYFP mutant was isolated (1,771 bp). C, Fluorescence values are the means ± se of four biological replicates, where each replicate represents the median measurements of 10,000 cells.

Figure 5.

Expression levels of cyanobacterial promoters in Synechocystis and UTEX 2973. A, Structure of the cyanobacterial promoters adapted for the CyanoGate kit. Regions of P_cpc560 shown are the TFBSs (−556 to −381 bp), middle region (−380 to −181 bp), and the downstream TFBSs, RBS, and spacer (−180 to −5 bp). B, Expression levels of eYFP driven by promoters in Synechocystis and UTEX 2973 calculated from measurements taken from 10,000 individual cells. Values are the means ± se from at least four biological replicates after 48 h of growth (average OD₇₅₀ values for Synechocystis and UTEX 2973 cultures were 3.5 ± 0.2 and 3.6 ± 0.2, respectively). See Supplemental Figure S2 for more info.

Figure 6.

Expression levels of heterologous and synthetic promoters in Synechocystis and UTEX 2973. A, Structure and alignment of eight new synthetic promoters derived from the BioBricks BBa_J23119 library and P_trc10 promoter design (Huang et al., 2010). B, Expression levels of eYFP driven by promoters in Synechocystis and UTEX 2973 calculated from measurements taken from 10,000 individual cells. C, Correlation analysis of expression levels of synthetic promoters tested in Synechocystis and UTEX 2973. The coefficient of determination (R²) is shown for the J23119 library (red), new synthetic promoters (pink), and trc variants (dark red). Values are the means ± se from at least four biological replicates after 48 h of growth (average OD₇₅₀ values for Synechocystis and UTEX 2973 cultures were 3.5 ± 0.2 and 3.6 ± 0.2, respectively). See Supplemental Figure S2 for more info.

Figure 7.

Protein expression levels in Synechocystis and UTEX 2973 cells. A, Confocal images of wild-type (WT) strains and mutants expressing eYFP (eYFP fluorescence shown in green on bright field images) driven by the J23119 promoter (bars = 10 µm). B, Representative immunoblot of protein extracts (3 µg protein) from mutants with different promoter expression cassettes (as in Fig. 6) probed with an antibody against eYFP. The protein ladder band corresponds to 30 kD. C, Relative eYFP protein abundance relative to that in UTEX 2973 mutants carrying the J23119 expression cassette. D to F, Cell density (D), protein content per cell (E), and protein density per estimated cell volume (F) for Synechocystis and UTEX 2973. Asterisks (*) indicate significant difference (P < 0.05) as determined by Student’s t test. Values are the means ± se of four biological replicates.

Figure 8.

Cell growth and expression levels of eYFP with the RK2 replicative origin in Synechocystis. A, Growth of strains carrying RK2 (vector pSEVA421-T-eYFP), RSF1010 (pPMQAK1-T-eYFP), or empty pPMQAK1-T, with cultures containing appropriate antibiotic selection. Growth was measured as OD₇₅₀ under a constant illumination of 100 µmol photons m⁻² s⁻¹ at 30°C. B, Expression levels of eYFP after 48 h of growth calculated from measurements taken from 10,000 individual cells. C, Plasmid copy numbers per cell after 48 h of growth. Lowercase letters indicating significant difference (P < 0.05) are shown, as determined by ANOVA followed by Tukey’s honestly significant difference tests. Values are the means ± se of four biological replicates.

Figure 9.

Gene regulation system using CRISPRi in Synechocystis. A, Four target regions were chosen as sgRNA protospacers to repress eYFP expression in Olive-eYFP (Fig. 4): “CCAGGATGGGCACCACCC” (+31), “ACT​TCA​GGG​TCA​GCT​TGC​CGT” (+118), “AGG​TGG​TCA​CGA​GGG​TGG​GCC​A” (+171), and “AGA​AGT​CGT​GCT​GCT​TCA​TG” (+233). B, eYFP fluorescence of Olive-eYFP expressing constructs carrying sgRNAs with and without dCas9 (representative of 10,000 individual cells). Untransformed Olive-eYFP and the Olive mutant were used as controls. Lowercase letters indicating significant difference (P < 0.05) are shown, as determined by ANOVA followed by Tukey’s honestly significant difference tests. Values are the means ± se of four biological replicates.