Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria

• Published in: Microorganisms (Basel) Vol. 7; no. 8; p. 263
• Main Authors: Kelly, Ciarán L, Taylor, George M, Šatkutė, Aistė, Dekker, Linda, Heap, John T
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 16.08.2019; MDPI
• Subjects: Binding sites; Carbon dioxide; Cyanobacteria; E coli; Flow cytometry; Gene expression; Genes; Genetics; Genomes; Glucose; Heterotrophic organisms; inducible expression; Operons; Plasmids; Rhamnose; RNA polymerase; Synechocystis; synthetic biology; Transcription; transcriptional terminators; United States > US; synthetic biology; Synechocystis; inducible expression; transcriptional terminators; cyanobacteria
• ISSN: 2076-2607; 2076-2607
• DOI: 10.3390/microorganisms7080263

Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and to 'insulate' genes and operons from one another. Insulating gene expression can be particularly important when heterologous or synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally-integrated, rhamnose-inducible expression system from hypothesised 'read-through' from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.