Inducible antisense RNA tool for differential gene expression in cyanobacteria

Cyanobacteria have emerged as a promising platform for sustainable methods of chemical and fuel production, but their utility is reduced by a lack of functional metabolic engineering tools, particularly, tools for predictable control of gene expression. The goal of our research is to develop methods for differential expression of native and heterologous genes in the cyanobacterium Synechococcus sp. PCC 7002. Synthetic biology was used to modify a native cyanobacterial promoter for anhydrotetracycline-based induction, an asRNA system was adapted for use in PCC 7002, and we aim to investigate the role of Hfq in asRNA function. An anhydrotetracycline (aTc) dependent induction system was constructed that allows for variable control of gene expression, with the current construct exhibiting 29-fold induction with 1 μg/mL aTc. This aTc dependent induction system will be used to control expression of trans-acting antisense RNAs (asRNA). asRNA functions by binding to the ribosome binding site of the mRNA transcript and inhibiting translation, providing a method to fine-tune gene expression and turn down native pathways. The IS10 asRNA module from E. coli was adapted into PCC 7002, demonstrated as functional, and improved by increasing the ratio of asRNA to the target mRNA. Additionally, the role of a small RNA chaperone, Hfq, is being investigated in RNA stability and asRNA function in PCC 7002. These tools and basic science knowledge will increase the utility of cyanobacteria in developing sustainable methods for chemical and fuel production.