Transformation of the cyanobacteriumSynechocystissp. PCC 6803 as a tool for genetic mapping: optimization of efficiency

Abstract

The cyanobacterium Synechocystis sp. PCC 6803 is transformable at high efficiency and integrates DNA by homologous double recombination. However, several genetic mapping procedures depend on the ability to generate transformants even with very small amounts of added DNA. This study is aimed at optimizing the transformation efficiency at limiting concentrations of exogenous DNA. The transformation efficiency showed little sensitivity to experimental conditions. Transformation with circular plasmid DNA was found to be no more than 30% more efficient than with linearized plasmid DNA. The efficiency of transformation remained essentially the same in the presence of competing DNA, indicating that the capacity of DNA uptake by the cells is not limiting. The incubation time of cells with DNA before plating (0-8 h) affected the transformation efficiency by up to 3-fold. Only minor changes in the efficiency were observed as a function of the presence of a membrane filter on the plate or the presence of TAE or TBE gel buffer residues in the transformation mixture. However, transformability of the host strain of Synechocystis sp. PCC 6803 was increased by two orders of magnitude if the sll1354 gene encoding the exonuclease RecJ was deleted. Therefore, the transformation efficiency of Synechocystis sp. PCC 6803 with exogenous DNA appears to be determined primarily by intracellular processes such as the efficiency of DNA processing and homologous recombination.

Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects

“…Over the past decade, Synechocystis 6803 metabolism has been modeled [69,70,71,72,73,74,75,76,77,78,79] to understand and improve cyanobacterial biofuel production. In 2009, the first genome-scale model of Synechocystis 6803 to predict the effect of biofuel production was produced [80].…”

“…There are many factors that affect the frequency and efficiency of transformation in cyanobacteria, which can vary between 10 −3 and 10 −8 [4]. These include the used strain, the length, form and concentration of used DNA and also the method used for transformation [72]. The optimum conditions for transformation of the model organism Synechocystis sp.…”

“…The optimum conditions for transformation of the model organism Synechocystis sp. PCC 6803 are well described [72,73]. …”

“…[70,73,74]. It was noted that, in Synechocystis 6803, exogenous DNA is integrated into the genome following natural transformation by means of a well-established double homologous recombination system [72,73]. Cyanobacteria that use systems with homologous recombination mechanisms allow for more effective gene modifications to wild-type genes [9].…”

Dark Anaerobic Hydrogen Production in the Mutants of Synechocystis sp. Strain PCC6803-GT Defective in Lactate Dehydrogenase Activity and/or Alcohol Dehydrogenase Activity, Incubated in Buffer Solutions With or Without Glucose

“…strain PCC6803 (GT strain) is transformable and represents both photoautotrophic growth on carbon dioxide and heterotrophic growth on glucose (Rippka et al, 1979;Kufryk et al, 2002). During phototrophic growth, carbon dioxide is incorporated into ribulose bisphosphate (RuBP) in the Calvin cycle to form 3-P-glycerate that is further converted to glucose in glycogen for storage utilizing the ATP and NADPH produced by the light-dependent reactions.…”