Improved genetic transformation of Synechococcus elongatus PCC 7942 using linear DNA fragments in association with a DNase inhibitor
Daniela Volcan Almeida, Stefani Betina Boschmann Martens, Carlos Frederico Ceccon Lanes, Luis Fernando Marins
Abstract
The genetic manipulation in many cyanobacterial strains is challenging yet. Thus, the development of new transformation protocols is desirable to facilitate the genetic engineering in cyanobacteria. Transformations using linear fragments yielded by PCR have advantages such as: less laborious methodology, faster procedure, low cost and unnecessary cloning steps. However, some strains presence extracellular nucleases, which reduce the efficiency in obtaining transformants. In this study, we demonstrate an improved protocol for genetic transformation in Synechococcus elongatus PCC 7942 using linear fragments employing EDTA-mediated inhibition of DNases. To conduct the transformation, linear PCR products containing the spectinomycin antibiotic resistance gene were employed. As result, 40 mM EDTA treatment increased the number of transformants obtained by eightfold in comparison to the conventional protocol using plasmid DNA. Thus, the application of exonuclease inhibitors can be considered a relevant improvement to manipulate cyanobacteria in a more efficient, faster way and as a low-cost alternative. This protocol must be helpful for other strains of cyanobacteria.
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References
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