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Title: Tunable riboregulator switches for post-transcriptional control of gene expression

The most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme catalyst. Until recently engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or by using one of several strategies to knock down or knock out a wasteful gene. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. We report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. Our design includes a cis-repressor at the 5’ end of the mRNA that forms a stem-loop helix occluding the ribosome binding site and blocking translation. An activating-RNA, expressed in trans, frees the RBS turning on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability followed by directed evolution on a portion of each trans-activator to fine tune translation. We report a cis-repressor that can completely shut off translation of antibiotic resistance reporters and a trans-activator that restores translation. We have shown it is possible to use riboregulators to achieve translational control of gene expression over a widemore » dynamic range. Using a bioluminescent reporter system, we demonstrated an ON/OFF ratio >300. We have demonstrated that a targeting sequence can be changed to develop riboregulators that can independently regulate translation of many genes with minimal cross-talk. In a SELEX experiment, we demonstrated that by subtly altering the sequence of the trans-activator, it is possible to alter the equilibrium between repressed and activated states and achieve intermediate translational control.« less
 [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [1]
  1. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Bioenergy and Biome Sciences, Bioscience Division
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Theoretical Biology and Biophysics, Theoretical Division
Publication Date:
Report Number(s):
Journal ID: ISSN 2161-5063
Grant/Contract Number:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 4; Journal Issue: 12; Journal ID: ISSN 2161-5063
American Chemical Society
Research Org:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org:
USDOE Laboratory Directed Research and Development (LDRD) Program
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; Biological Science; translation; gene expression; pathway engineering; riboregulators; synthetic biology; translational control
OSTI Identifier: