Fig. 1: Diagram of the REEP genetic engineering system for cyanophages.

a The process of producing recombinant-containing phage lysates. A recombination template is composed of homologous regions (H1, H2, 200–300 bp each) that flank the target gene to be deleted in the phage genome and a short TAG sequence that will be inserted in place of the target gene. This is cloned into the pREC plasmid and inserted into Synechococcus to produce the recombination host. This host is infected with wild-type cyanophages. Homologous recombination that occurs during infection produces a lysate containing wild-type (black) and recombinant (red) phages. b Enrichment and screening for the isolation of recombinant phages. (i) Multiple 96-well plates are infected with the recombinant-containing lysate at the optimal enrichment dilution, which is 5–10 phages per well. (ii) Once lysis is complete, the plates are screened by recombinant-specific PCR to detect recombinant phages based on their TAG sequence. Wells in which a recombinant phage was present among the initial 5–10 phages used for infection will now contain a lysate that is highly enriched with recombinants (>100-fold) and will be detected in the PCR screen. (iii) The highly enriched lysates from PCR positive wells are plated for plaque screening and isolation of the recombinant phage.