Transformation Techniques

Ease in the genetic modification process has been a very essential necessity for a cyanobacterial strain development using synthetic biology tools for metabolic engineering applications. Currently, three procedures are being widely used for the insertion of target gene into cyanobacterial host cells: transformation, conjugation and electroporation (Vioque, 2007). The efficiency of transformation in cyanobacteria depends on biochemical and physical barriers which varies from species to species (Stucken et al., 2013). Transformation also depends on the size, structural organization and concentration of the target DNA used in engineering process (Nagarajan et al., 2011). Transformation can be done by using either integrative plasmid or by replicative plasmid. DNA transfer by integrative plasmid employs the foreign DNA incorporation into the genomic DNA of host cells by the process called homologous recombination (Heidorn et al., 2011). Whereas, replicative plasmids replicate and express independently along with foreign DNA in the host cell (Wang et al., 2013). These two types of plasmids have been well developed for the transformation of cyanobacteria. It has already been demonstrated the possibility of using linear DNA segment in Synechocystis sp. for metabolic engineering applications (Nagarajan et al., 2011). Synechococcus elongatusPCC 7942 was first time engineered with linear DNA fragment using EDTA as DNases inhibitor (Daneilla et al., 2017). Some cyanobacterial strains like Synechocystis 6803 (Lindberg et al., 2010),Synechococcus PCC 7942 (Johnsberg et al., 2007) andSynechococcus PCC 7002 (Xu et al., 2011) are naturally competent to take foreign DNA. This attribute of natural competency is not common in other strains. Target DNA can be transferred in non-competent strains by a well-developed method called tri-parental conjugation, which employs helper, conjugal and replicable plasmids (Yu et al., 2015). DNA transmission from E. coli to nitrogen fixing cyanobacterial strains of Nostoc and Anabaena have been genetically manipulated (Ruffing, 2011). In a study Yu et al. (2015) has successfully applied tri-parental conjugation process inSynechococcus UTEX 2973 with the help of helper and conjugal plasmids because they are not naturally transformable, like its close relative Synechococcus PCC 6301. Table 5 summarizes the different DNA transfer strategies used for cyanobacterial genetic manipulation.
<Table 5 >