Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. Issue 2 (3rd September 2015)

• Record Type: Journal Article
• Authors: Zess, Erin K.; Begemann, Matthew B.; Pfleger, Brian F.
• Is Part Of: Biotechnology and bioengineering. Volume 113:Issue 2(2016)
• Page Start: 424
• Page End: 432
• Publication Date: 2015-09-03
• Subjects: Hfq -- cyanobacteria -- synthetic biology -- promoter -- induction system -- sRNA system -- RNA‐IN‐OUT; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.25713
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: Predictive control of gene expression is an essential tool for developing synthetic biological systems. The current toolbox for controlling gene expression in cyanobacteria is a barrier to more in‐depth genetic analysis and manipulation. Towards relieving this bottleneck, this work describes the use of synthetic biology to construct an anhydrotetracycline‐based induction system and adapt a trans‐acting small RNA (sRNA) system for use in the cyanobacterium Synechococcus sp. strain PCC 7002. An anhydrotetracycline‐inducible promoter was developed to maximize intrinsic strength and dynamic range. The resulting construct, PEZ tet, exhibited tight repression and a maximum 32‐fold induction upon addition of anhydrotetracycline. Additionally, a sRNA system based on the Escherichia coli IS10 RNA‐IN/OUT regulator was adapted for use in Synechococcus sp. strain PCC 7002. This system exhibited 70% attenuation of target gene expression, providing a demonstration of the use of sRNAs for differential gene expression in cyanobacteria. These systems were combined to produce an inducible sRNA system, which demonstrated 59% attenuation of target gene expression. Lastly, the role of Hfq, a critical component of sRNA systems in E. coli, was investigated. Genetic studies showed that the Hfq homolog in Synechococcus sp. strain PCC 7002 did not impact repression by the engineered sRNA system. In summary, this work describes new synthetic biology tools that can be applied to physiological studies, metabolic engineering, or sRNA platforms in Synechococcus sp. strain PCC 7002. Biotechnol. Bioeng. 2016;113: 424–432. © 2015 Wiley Periodicals, Inc. Abstract : New regulatory elements were engineered for controlling gene expression in the fast‐growing cyanobacterium, Synechococcus sp. strain PCC7002. Anhydrotetracycline inducible promoters were created by optimizing the location of TetR binding sites proximal to strong constitutive promoters resulting in a nearly 30‐fold dynamic range of YFP expression.