A promoter-probe vector-host system for the cyanobacterium, Synechocystis PCC6803 (original) (raw)
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Frontiers in Microbiology
To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.
Journal of Microbiological Methods, 2000
Two promoter probe vectors were constructed for the cyanobacterium Synechocystis sp. strain PCC 6803 using reporter genes, which can be easily detected and quantified in vivo by the ability of their encoded proteins to emit light. The vectors allow the transcriptional fusion of promoter sequences with the gfp and luxAB genes, respectively, and their stable integration into a neutral site of the Synechocystis chromosome. Functionality of these vectors was demonstrated by cloning the promoter of the isiAB operon into both promoter probe vectors and analyzing the stress-dependent emission of light by the obtained reporter strains. As was found before for the isiAB operon, the P reporter gene fusions were induced by iron isi AB starvation and high salt stress. Induction rates of mRNA of the wild type operon and the reporter gene fusions were found to be essentially the same, indicating that a promoter fragment containing all necessary regulatory elements has been cloned. However, using the gfp gene a slow increase of protein and fluorescence was found, while the luxAB reporter gene constructs led to a rapid increase in luminescence. The same was found after retransfer of cells back into control media, in which the Gfp protein disappeared slowly, while the LuxAB-based luminescence decreased rapidly. These experiments show that both reporter genes can be used in Synechocystis: the luxAB system seems to be favourable regarding reaction time, while the gfp system has the advantage of being independent from any substrate.
Synthetic Biology, 2018
Cyanobacteria are promising ‘low-cost’ cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the ‘green’ Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to...
A Novel Expression Vector for the Cyanobacterium, Synechococcus PCC 6301
DNA Research, 1994
A cyanobacterial expression vector was constructed using ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO) promoter and terminator sequences derived from Synechococcus PCC 6301. The recombinant plasmid, designated pARUB19, has an ampicillin-resistant (Ap R) gene as a selectable marker and four unique restriction sites to allow the insertion of foreign genes. Using this vector, the luciferase gene from the firefly, Photinus pyralis, was introduced into Synechococcus PCC 6301 cells. The luciferase expression vector could be maintained stably in the host cells. Light production of luciferin/luciferase was detected in the transformants. Luciferase amounted to 1.2% of the total soluble protein. This plasmid may facilitate higher levels of foreign gene expression in Synechococcus PCC 6301.
Journal of Advances in Biology & Biotechnology
Photosynthetic Cyanobacteria can be used as a chassis for different synthetic biology approaches. However, quantitative comparison of tools for engineering, such as those for heterologous gene expression, is often not available. Here, we report the comparative quantification of heterologous protein production in Synechococcussp.PCC 7002 regarding protein expression cassettes and locations of foreign gene integration using sf-GFP as a reporter. We used promoter cpc560 as reference because it was described as a "super strong" promoter. sf-GFP-expression constructs were integrated into neutral sites NS_1, NS_2, NS_3 and the extrachromosomal plasmid pAQ1. The latter induced a sf-GFP level of approximately 10-fold in comparison to a reference promotor expression. Protein-fusion with 6xHis increased sf-GFP as well as expression of sf-GFP fusion with ß subunit of phycocyanin.
Nucleic Acids Research, 1984
The putative promoter region of the chloroplast encoded ps2B gene (the gene encoding the 32kD herbicide binding B protein of photosystem II (1-4)) has been fused to a chloramphenicol acetyl transferase (CAT) gene that lacks Its bacterial promoter and found to accurately Initiate transcription from this promoter when Introduced Into the cyanobacterium, Anacystls nldulans R2 (or Into E. coll). The chloroplast promoter-CAT fusion was Introduced Into the cells on a plasmid that contains plasrald replication origins for E. coll and Anacystls as well as a second antibiotic resistance marker. Cells transformed with corresponding vectors lacking the promoter region do not express CAT.
A facile method to study DNA synthesis in the cyanobacterium Synechocystis PCC 6803
Current Microbiology, 1994
For the efficient study of replication of DNA, the cyanobacteriumSynechocystis PCC 6803 was first permeabilized by eitherl-α-lysophosphatidylcholine (LPC) or lysozyme-EDTA treatment. Permeability of the treated cells was evidenced by the incorporation of exogenously added32P-TTP into DNA. In cells permeabilized by treatment with either method, the32P-TTP incorporation at 30°C was appreciably higher than that in untreated control cells and increased with time for about 4 h. In addition, treated cells became permeable to proteins such as DNase I and micrococcal nuclease, which entered cells and degraded the newly synthesized DNA. Lysozyme/EDTA-treated cells not only incorporated32P-TTP more efficiently than did LPC-permeabilized cells, but were capable of uptake and synthesis of exogenously supplied cyanobacterial plasmids isolated fromSynechocystis 6803. This capacity of lysozyme/EDTA-treatedSynechocystis to catalyze replication of exogenous DNA will allow the facile identification of DNA replication origins and their related regulatory sequences.
Current Microbiology, 1994
An expression vector, pFC1, has been constructed based on the promiscuous plasmid RSF1010, which provides autonomous replication in several cyanobacteria of the genera Synechocystis and Synechococcus . pFC1 harbors the hci857 repressor-encoding gene andpR promoter, followed by the hcro ribosome-binding site and ATG translation initiation codon. The latter is located within the unique NdeI restriction site (CATATG) of pFC1 and can be exposed after cleavage with this enzyme for in-frame fusion with the protein-coding sequence to be expressed. The Escherichia coil lacZ reporter gene cloned in pFC1 appeared to be highly expressed in heat-induced E. coil or cyanobacterial cells. In every case, [3-galactosidase amounted to at least 10% of soluble proteins.
Synthetic Biology, 2021
Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively...