Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology

Abstract

Cyanobacteria are suitable for sustainable, solar-powered biotechnological applications. Synthetic biology connects biology with computational design and an engineering perspective, but requires efficient tools and information about the function of biological parts and systems. To enable the development of cyanobacterial Synthetic Biology, several molecular tools were developed and characterized: (i) a broad-host-range BioBrick shuttle vector, pPMQAK1, was constructed and confirmed to replicate in Escherichia coli and three different cyanobacterial strains. (ii) The fluorescent proteins Cerulean, GFPmut3B and EYFP have been demonstrated to work as reporter proteins in cyanobacteria, in spite of the strong background of photosynthetic pigments. (iii) Several promoters, like P(rnpB) and variants of P(rbcL), and a version of the promoter P(trc) with two operators for enhanced repression, were developed and characterized in Synechocystis sp. strain PCC6803. (iv) It was shown that a system for targeted protein degradation, which is needed to enable dynamic expression studies, is working in Synechocystis sp. strain PCC6803. The pPMQAK1 shuttle vector allows the use of the growing numbers of BioBrick parts in many prokaryotes, and the other tools herein implemented facilitate the development of new parts and systems in cyanobacteria.

Figure 1.

Construction of the broad-host-range BioBrick shuttle vector pPMQAK1. The shaded 3318-bp DNA fragment of pSB1AK3-BBa_B0015, which includes the BioBrick cloning site containing part BBa_B0015 flanked by terminators and cassettes conferring resistance against ampicillin and kanamycin, was amplified with PCR using primers pSB03-KpnI-f and pSB03-MunI-r (Table 1). The shaded 9281-bp DNA fragment of pAWG1.1, which includes the replicon derived from RSF1010, was amplified with PCR using primers pAWG02-MunI-f and pAWG02-KpnI-r (Table 1). Intermediate plasmid pPMQAK1-BBa_B0015 (data not shown) was formed by digesting the two DNA fragments with KpnI and MunI and joining the resulting fragments. Finally, BioBrick part BBa_B0015 was exchanged with part BBa_P1010 to produce the pPMQAK1-BBa_P1010 plasmid. Abbreviations: BioBrick (BioBrick cloning site), T (double transcriptional terminator BBa_B0015), AmpR (ampicillin resistance cassette), KmR (kanamycin resistance cassette), CmR (chloramphenicol resistance cassette), RepA, RepB and RepC (replication proteins A, B and C), MobA, MobB and MobC (mobilization proteins A, B and C), OriV (vegetative origin of replication), GFP (green fluorescent protein), P_petE (petE promoter) and S (ligation site of the two DNA fragments).

Figure 2.

Construction of the rbcL promoter variants and the structure of P_trc1O and P_trc2O. (A) The upstream region (–277 to –1, relative to the ATG) of the rbcL gene in Synechocystis sp. strain PCC 6803. a: predicted NtcA-binding site (57), b: putative –10 element (57), c: putative –35 element (56), d: –10 element (56), e: putative ribosome-binding site (RBS) (56), f: start codon ATG of the rbcL gene. The variants ‘1’ (P_rbcL1A, P_rbcL1B, P_rbcL1C) lack the predicted NtcA-binding site and the AT-rich region upstream (–277 to –215, relative to the ATG of the rbcL gene), whereas in the variants ‘2’ (P_rbcL2A, P_rbcL2B, P_rbcL2C) it is present. In the variants ‘A’ (P_rbcL1A, P_rbcL2A) the BioBrick RBS (black box), replacing the native RBS, was introduced on the primer, whereas in the variants ‘B’ (P_rbcL1B, P_rbcL2B) the BioBrick RBS was attached by standard assembly, resulting in an additional 8-bp scar (TACTAGAG, gray box). The variants ‘C’ (P_rbcL1C, P_rbcL2C) lack part of the 3′-end (–48 to –18, relative to the ATG of the rbcL gene), and the RBS (black box) is attached by standard assembly, resulting in an additional 8-bp scar (TACTAGAG, grey box) as in the variants ‘B’. (B) Alignment of the rbcL promoter variants ‘A’, ‘B’ and ‘C’ at the 3′-end. The first 48 bp of the rbcL gene upstream of the ATG and the consensus Sll1594 (NdhR) DNA-binding motif TCAATG(N10)ATCAAT are shown as references. The consensus Sll1594 (NdhR) DNA-binding motif is underlined, the consensus LysR-type motif T(N11)A in black boxes, the putative native RBS and the BioBrick RBS in bold/italic, the 8-bp scar in gray boxes, and the start codon ATG boxed. (C) The structure of P_trc1O and P_trc2O. In P_trc1O, the –35 (TTGACA) and –10 (TATAAT) elements are divided by a 17-bp spacer. The 21 bp O1 lac operator is located 6-bp downstream of the –10 element. In P_trc2O, the 20 bp ideal Oid lac operator is located 34-bp upstream of the –35 element. The distance between the middle of the two operators is 88 bp.

Figure 3.

Replicative ability of pPMQAK1 in cyanobacteria. To determine the ability of pPMQAK1 for replication in the cyanobacteria Synechocystis sp. strain PCC 6803, Nostoc sp. strain PCC 7120 and N. punctiforme strain ATCC 29133, a P_trc1O-GFP (GFP) reporter construct was inserted into the vector which was subsequently transferred to the cyanobacterial cells by the means of triparental mating. GFP: GFPmut3B fluorescence of wild-type cultures or cultures containing the pPMQAK1-GFP reporter constructs. DIC + Red: Control, red autofluorescence stemming from phycobilisomes and photosystem II complexes superimposed with a transmission image in DIC mode. Abbreviations: WT, wild-type; GFP, green fluorescent protein; DIC, differential interference contrast; Red, red auto-fluorescence.

Figure 4.

Comparison of excitation and emission spectra of the fluorescent proteins Cerulean, GFPmut3B and EYFP in cyanobacterial background. Background-subtracted excitation (solid line) and emission (dotted line) spectra of the fluorescent proteins Cerulean (blue), GFPmut3B (green) and EYFP (black) expressed in Synechocystis sp. strain PCC 6803.

Figure 5.

Specific activities of the six rbcL promoter variants and confirmation of fluorescence measurements by SDS–PAGE/western blot analysis (A) Specific activities of the six rbcL promoter variants in comparison to P_rnpB and P_trc1O. The activities were measured by means of GFPmut3B fluorescence and divided by the absorbance of the cultures at 750 nm. The data represent mean ± SD of triple measurements of three independent cultivations. (B) Coomassie-stained SDS–PAGE of total proteins extracted from Synechocystis sp. strain PCC 6803 cultures that expressed the GFPmut3B reporter constructs of the six rbcL promoter variants, P_rnpB and P_trc1O, respectively. (C) Western blot analysis of the same samples as in (B) using anti-GFP, N-terminal, antibodies.

Figure 6.

Specific promoter activities of P_trc1O, P_lac, P_tet, P_R and P_rnpB in E. coli DH5α (white bar) and Synechocystis sp. strain PCC 6803 cells (black bar). The activities were measured by means of GFPmut3B fluorescence and divided by the absorbance of the cultures at 595 nm (E. coli) or 750 nm (Synechocystis), respectively. The data represent mean ± SD of triple measurements of three independent cultivations.

Figure 7.

Specific promoter activities of P_trc1O and P_trc2O in E. coli DH5α without (–) and with ( + ) the expressed lac repressor under non-induced (black bars) and induced (gray bars) conditions. The activities were measured by means of GFPmut3B fluorescence and divided by the absorbance of the cultures at 595 nm. The data represent mean ± SD of triple measurements of three independent cultivations. The lac repressor was expressed on the same plasmid as the GFPmut3B reporter constructs under the control of P_rnpB. Induction was done by 1 mM IPTG.

Figure 8.

Specific promoter activities of P_trc1O and P_trc2O in Synechocystis sp. strain PCC 6803 without (–) and with ( + ) the expressed lac repressor under non-induced (black bars) and induced (gray bars) conditions. The activities were measured by means of GFPmut3B fluorescence and divided by the absorbance of the cultures at 750 nm. The data represent mean ± SD of triple measurements of three independent cultivations. The lac repressor was expressed on the same plasmid as the GFPmut3B reporter constructs under the control of P_rnpB or P_rbcL2A (as indicated). The specific promoter activities of P_rnpB and P_rbcL2A GFPmut3B reporter constructs are shown as indication for the lac repressor levels in the cells. Induction was done by 2 mM IPTG.

Figure 9.

Specific promoter activities of P_trc1O (black bars) and P_trc2O (light and dark grey bars) in Synechocystis sp. strain PCC 6803 against the IPTG concentration. The activities were measured by means of GFPmut3B fluorescence and divided by the absorbance of the cultures at 750 nm. The data represent mean ± SD of triple measurements of three independent cultivations. The lac repressor was expressed on the same plasmid as the GFPmut3B reporter constructs under the control of P_rnpB (black and light grey bars) or P_rbcL2A (dark grey bars).

Figure 10.

Specific fluorescence intensities of EYFP and its three different degradation tagged variants in Synechocystis sp. strain PCC 6803. The EYFP fluorescence was measured after 48-h cultivation time and divided by the absorbance of the cultures at 750 nm. The data represent mean ± SD of triple measurements of three independent cultivations. EYFP (BBa_E0030) or the degradation tagged variants EYFP-ASV (BBa_E0036), EYFP-AAV (BBa_E0034) and EYFP-LVA (BBa_E0032) were expressed by the promoter P_trc1O.