Riboswitch Signal Amplification by Controlling Plasmid Copy Number.

Riboswitches are cis-acting RNA devices in mRNAs that control gene expression in response to chemical inputs. As RNA aptamers that recognize diverse classes of molecules can be isolated by in vitro selection, synthetic riboswitches hold promise for various applications in synthetic biology. One of the major drawbacks of riboswitches, however, is their limited dynamic range. A high level of gene expression in the OFF state (leakage) is also a common problem. To address these challenges, we designed and constructed a dual-riboswitch plasmid in which two genes are controlled by theophylline-activated riboswitches. One riboswitch controls the gene of interest, and another riboswitch controls RepL, a phage-derived replication protein that regulates the plasmid copy number. This single-plasmid system afforded an ON/OFF ratio as high as 3900. Furthermore, we used the system to control CRISPR interference (CRISPRi) targeting endogenous genes, and successfully observed expected phenotypic changes in Escherichia coli.

[1]  Y. Yokobayashi,et al.  Engineering artificial small RNAs for conditional gene silencing in Escherichia coli. , 2012, ACS synthetic biology.

[2]  R. Breaker.  Riboswitches and the RNA world. , 2012, Cold Spring Harbor perspectives in biology.

[3]  W. Mahoney,et al.  Primers with 5' flaps improve real-time PCR. , 2007, BioTechniques.

[4]  Navjot Singh,et al.  Translational control and Rho-dependent transcription termination are intimately linked in riboswitch regulation , 2017, Nucleic acids research.

[5]  R. Batey,et al.  Modularity of select riboswitch expression platforms enables facile engineering of novel genetic regulatory devices. , 2013, ACS synthetic biology.

[6]  Yohei Yokobayashi,et al.  Controlling Mammalian Gene Expression by Allosteric Hepatitis Delta Virus Ribozymes , 2013, ACS synthetic biology.

[7]  E. Newman,et al.  An Escherichia coli Mutant That Makes Exceptionally Long Cells , 2015, Journal of bacteriology.

[8]  Harris H. Wang,et al.  Multiplex recording of cellular events over time on CRISPR biological tape , 2017, Science.

[9]  Atsushi Ogawa,et al.  An Artificial Aptazyme‐Based Riboswitch and its Cascading System in E. coli , 2008, Chembiochem : a European journal of chemical biology.

[10]  G. Fleet,et al.  Method for the isolation of highly purified Salmonella flagellins , 1985, Journal of clinical microbiology.

[11]  Feng Zhang,et al.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system , 2013, Nucleic acids research.

[12]  Yohei Yokobayashi,et al.  An efficient platform for genetic selection and screening of gene switches in Escherichia coli , 2009, Nucleic acids research.

[13]  Mark S Dunstan,et al.  Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species. , 2014, Journal of the American Chemical Society.

[14]  B. Strukelj,et al.  Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes , 2008, Microbial cell factories.

[15]  H. Erickson,et al.  In Vivo Characterization of Escherichia coli ftsZ Mutants: Effects on Z-Ring Structure and Function , 2003, Journal of bacteriology.

[16]  Ronny Lorenz,et al.  Design criteria for synthetic riboswitches acting on transcription , 2015, RNA biology.

[17]  H. Salis,et al.  Automated physics-based design of synthetic riboswitches from diverse RNA aptamers , 2015, Nucleic acids research.

[18]  F. Blattner,et al.  Genome of Bacteriophage P1 , 2004, Journal of bacteriology.

[19]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[20]  Markus Wieland,et al.  Improved aptazyme design and in vivo screening enable riboswitching in bacteria. , 2008, Angewandte Chemie.

[21]  S. Harbaugh,et al.  Amplifying Riboswitch Signal Output Using Cellular Wiring. , 2017, ACS synthetic biology.

[22]  Mario Mörl,et al.  Synthetic Riboswitches: From Plug and Pray toward Plug and Play. , 2017, Biochemistry.

[23]  Florian Groher,et al.  Synthetic riboswitches - A tool comes of age. , 2014, Biochimica et biophysica acta.

[24]  H. Salis,et al.  Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites , 2013, Nucleic acids research.

[25]  David G. E. Smith,et al.  Escherichia coli O 157 : H 7 Colonization in Cattle following Systemic and Mucosal Immunization with Purified H 7 Flagellin , 2008 .

[26]  S. K. Desai,et al.  Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species , 2010, Applied and Environmental Microbiology.

[27]  Y. Yokobayashi,et al.  Controlling Bdellovibrio bacteriovorus Gene Expression and Predation Using Synthetic Riboswitches. , 2017, ACS synthetic biology.