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 >