Synthetic counter-selection markers and their application in genetic modification of Synechococcus elongatus UTEX2973

Due to its robustness to environmental stresses and fast growth, Synechococcus elongatus UTEX2973 is developed as a new model for researches on cyanobacterial molecular biology and biotechnology. However, systematic genetic modifications of S. elongatus UTEX2973 were hindered by the lack of effective genetic manipulation tools, especially available counter-selection markers. Here, six synthetic counter-selection markers (SCOMs) were assembled by fusing six toxin genes from either Escherichia coli or cyanobacteria with a theophylline-inducible promoter. The SCOMs containing SYNPCC7002_G0085 from Synechococcus sp. PCC7002 or mazF from E. coli were proved to be inducible by theophylline in S. elongatus UTEX2973. By using the mazF-based SCOM, the neutral locus 1 and 23 small regulatory RNAs were completely deleted from the genome of S. elongatus UTEX2973 after one round of selection with both kanamycin and theophylline. The genetic tools developed in this work will facilitate future researches on molecular genetics and synthetic biology in S. elongatus UTEX2973. KEY POINTS: • Two inducible counter-selection markers are lethal to S. elongatus UTEX2973. • The counter-selection marker benefits the gene targeting in S. elongatus UTEX2973. • Twentry-three small regulatory RNAs were fully deleted via the novel gene targeting method.