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A mutation is a change in the sequence of bases of DNA or RNA in a genome. Some mutations occur during replication of the genome due to errors made by the polymerase enzymes that replicate DNA or RNA. Unlike DNA polymerase, RNA polymerase is prone to errors because it is not capable of “proofreading” its work. Viruses with RNA-based genomes, like HIV, therefore accrue mutations faster than viruses with DNA-based genomes. Because mutation and recombination provide the raw material...
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Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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Updated: Jul 4, 2025

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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T7 phage-assisted evolution of riboswitches using error-prone replication and dual selection.

Eduardo Goicoechea Serrano1,2, Carlos Blázquez-Bondia1, Alfonso Jaramillo3,4

  • 1Warwick Integrative Synthetic Biology Centre and School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK.

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Summary

We developed T7AE, a novel phage evolution system, to efficiently select functional theophylline riboswitches. This method enriches gene switches for synthetic biology applications.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • RNA biology

Background:

  • Riboswitches are non-coding RNA sensors that regulate gene expression.
  • Selecting and evolving functional riboswitches is challenging.
  • Existing methods lack efficiency in enriching these genetic sensors.

Purpose of the Study:

  • To develop a novel phage-based system for the evolution of theophylline riboswitches.
  • To create a dual-selection method for enriching functional riboswitches in both ON and OFF states.
  • To demonstrate the efficacy of the T7AE system in evolving a library of riboswitch variants.

Main Methods:

  • Engineered bacteriophage T7 by replacing DNA polymerase with a theophylline riboswitch-controlled transcription factor.
  • Created two host environments with specific genes (cmk or pifA) for dual selection.
  • Applied the T7AE system to a library of 65,536 randomized riboswitch variants.

Main Results:

  • Successfully enriched functional theophylline riboswitches from a large library.
  • Demonstrated selection in both riboswitch ON (cmk host) and OFF (pifA host) states.
  • Observed enrichment of phages encoding riboswitches conferring a fitness advantage.

Conclusions:

  • The T7AE system provides a powerful new tool for evolving gene switches, including non-coding RNA-based sensors.
  • This technique advances synthetic biology by enabling the development of novel RNA-based regulatory elements.
  • The dual-selection mechanism offers a robust approach for selecting functional riboswitches.