Originating in an environment without available fixed carbon, cyanobacteria have evolved as versatile organisms, capable of producing a large variety of organic compounds from simple inorganic sources that can be directly used or transformed into a commercial product. When the desired molecule is not naturally produced, genes or entire pathways can be introduced through a variety of methods and product yields can be increased by driving cell metabolism toward the desired product. There are more than 3350 species of cyanobacteria already described, with hundreds avail­able in culture collections (Guiry and Guiry). To date, 87 cyanobacterial genomes have been sequenced and depos­ited in public databases but only a few strains have been used in genetic manipulation studies (Heidorn et al.,

2011) . Many molecular tools are currently available and genetic manipulation can be pursued through conjuga­tion, electroporation or natural transformation. These techniques are constantly being revised or optimized for each host species and sample protocols are available elsewhere (Heidorn et al., 2011). So far, no cyanophage able to perform transduction has been described, never­theless this technique is still the object of great interest (Koksharova and Wolk, 2002).

Natural transformation is an appealing feature found in some cyanobacterial strains, with two standing out as being frequently used in genetic manipulation studies, Synechocystis sp. PCC 6803 (Pasteur Culture Collection) and Synechococcus sp. PCC 7002 (Grigorieva and Shesta­kov, 1982). These two strains are of significant interest due to the high yield of mutants achieved through this technique, making it widely used for both pure and applied science, from plant physiology studies to meta­bolic engineering aiming for the commercial production of biomolecules.

The high frequency of transformants with natural transformation is intimately linked with the nature of the transferred genetic material, with chromosomal DNA reaching up to 100-fold more viable transformants than when replicative plasmids are used as the source of DNA (Golden and Sherman, 1983; Shestako and Khyen, 1970). In fact, this is true specifically for replicative plas­mids since most of the transformation efficiency is recovered when a suicide plasmid is used (Tsinoremas et al., 1994). Thus, it would seem that the final localiza­tion of the inserted DNA plays a key role in the transfor­mation efficiency. This is argued to be related to the postreplicative processing of chromosomal DNA together with a putative robust recombination mecha­nism in these species (Flores et al., 2008). Natural trans­formation has being reported to be associated with pilus-related genes (Yoshihara et al., 2001; Yura, 1999), a natural machinery putatively adapted to take up exog­enous DNA with such high efficiency that different artificial procedures intended to increase the transfor­mation yield fail to improve the frequency of viable mutants (Zang et al., 2007). Unfortunately, natural trans­formation is not widespread in the cyanobacterial phylum and many species require other techniques for the efficient introduction of exogenous DNA.

Electroporation was first demonstrated in Anabaena sp. (Thiel and Poo, 1989) and today has been optimized for many strains. It has been shown to be effective despite the low yield in many cases (Koksharova and Wolk,

2002) . Unlike what is observed for green algae (Kilian et al., 2011), the procedures and electric pulse settings are not very different from those used with other bacte­rial phyla (Heidorn et al., 2011). However, even though it can be an effective method, the ease of natural transfor­mation and the higher yield of conjugation have left electroporation behind as a choice for mutagenesis.

Conjugation is the most commonly used technique for genetic engineering in terms of the diverse species with which it can be used, and, with the filamentous N2 fixing (heterocyst forming) cyanobacteria, it is the only effective technique thus far described. With the advent of molecular biology, plasmids of cyanobacterial origin were actively sought with the intention of produc­ing shuttle vectors allowing their transfer from E. coli to Synechococcus (Golden and Sherman, 1983). Since then,

E. coli has been widely used for conjugation with many filamentous strains, such as Nostoc sp. and Anabaena sp., and single cell strains, like Synechococcus sp. and Synechocystis sp. Although incorporation of DNA into the chromosome of many strains has proved to be relatively easily achieved when using linear DNA or suicide plasmids, it has proved challenging to make cyanobacteria harbor replicative plasmids. During conjugation, the plasmid is relaxed and single-stranded DNA is driven to the recipient cell through the type four secretion system by the enzyme relaxase. Once in the recipient cell, the transferred DNA will have its anti­sense strand resynthesized and this newly reformed plasmid can integrate itself into the genome or autore­plicate. The vectors used in cyanobacteria must contain the replicons for both organisms, donor and recipient, a mobilization site (origin of transfer, e. g. bom, nic and oriT), a selective marker effective for both organisms, and a codon optimization to avoid the broad range of restriction enzymes harbored by cyanobacteria, which has been found to be an important hurdle to successful conjugation (Elhai et al., 1997; Flores et al., 2008; Wolk et al., 1984). Extra enzymes might be needed to ensure a successful transfer, which could be encoded on sec­ondary (aka helper) plasmids. Among these special enzymes are some endonucleases, intended to cut the cargo plasmid at the bom site and promote transfer, and methylases to protect the transferred DNA against the restriction enzymes in the recipient. Detailed proce­dures, strategies and strains used are amply reviewed elsewhere (Heidorn et al., 2011).