Synechococcus Engineering Kits
The GeneArt® Synechococcus Engineering Kits employ a cyanobacterium-based model system that offers a simplified approach for studying circadian rhythms, nutrient regulation, environmental response, lipid metabolism, and protein expression. Using the optimized cloning and expression vectors, frozen cells, specially formulated Gibco® medium, and easy-to-follow protocols, growth and transformation of the cyanobacterium Synechococcus elongatus is straightforward (Figure 1), which helps to ensure reliable results and faster scale-up.
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Characteristics of S. elongatus
Synechococcus elongatus is a cyanobacterium—one of the unicellular photosynthetic prokaryotes sometimes referred to as blue-green algae.
S. elongatus is an excellent model system with the following features:
- Fully sequenced and annotated genome (strain PCC 7942)
- Easily manipulated by transformation or conjugation from E. coli
- Small genome size, ~2.7 Mb
- Neutral cloning loci can be disrupted with no aberrant phenotype, allowing homologous recombination in the cell’s chromosome
- Can be engineered to produce and secrete high-value products (Appl Environ Microbiol 76:3462 (2010))
|Figure 1: Frozen S. elongatus cells were resuscitated in Gibco® BG-11 medium, in triplicate samples. Growth was monitored by OD750 measurements taken periodically during the study.|
S. elongatus Cloning Vectors
We have developed two S. elongatus expression vectors: pSyn_1, which contains a standard multiple cloning site (MCS), and pSyn_1/D-TOPO®, which allows you to carry out rapid and efficient directional cloning of a blunt-end PCR product using our proven TOPO® cloning technology.
Our S. elongatus vectors contain:
Transformation of S. elongatus
Transformation of S. elongatus relies on homologous recombination between the cell’s chromosome and exogenous, non-autonomously replicating DNA containing sequences homologous to the chromosome. Sites on the S. elongatus chromosome have been developed as cloning loci, called “neutral sites.” They can be disrupted without causing any aberrant phenotype, thus allowing the homologous recombination of ectopic sequences.
When S. elongatus is transformed with a vector containing an antibiotic resistance cassette and neutral site sequences, homologous recombination occurs between the neutral site vector and the S. elongatus chromosome; the transgene and the selective marker are inserted into the neutral site and the vector sequence is lost, allowing the expression of the introduced genes in S. elongatus.
Engineered S. elongatus Strains Deliver Improved Production
Our manufactured S. elongatus cell lots have very good transformation efficiencies, typically around 5 x 104 per microgram of DNA. To test the expression obtained using the GeneArt® Synechococcus Engineering Kit, we created a lactate-producing strain of S. elongatus. After 5 days, lactate production in the engineered strain was dramatically improved compared to the untransformed strain (Figure 2).
|Figure 2: Lactate production in S. elongatus engineered with GeneArt® Algae Engineering Kits. A strain of S. elongatus already expressing the ldh1 gene was transformed with the pSyn_1/D-TOPO® vector containing the ldhA gene. Transformants were selected on spectinomycin and grown in 4 mL of Gibco® BG-11 medium for 6 days. Lactate production was measured by an enzymatic assay. The data above are representative of production from a single wild type culture and a single engineered culture.|