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Ribozyme-Mediated Gene-Fragment Complementation for Nondestructive Reporting of DNA Transfer within Soil. | LitMetric

Ribozyme-Mediated Gene-Fragment Complementation for Nondestructive Reporting of DNA Transfer within Soil.

ACS Synth Biol

Department of BioSciences, Rice University, MS-140, 6100 Main Street, Houston, Texas 77005, United States.

Published: November 2024

AI Article Synopsis

  • Enzymes that produce volatile gases can be used to track microbial activities in soil, but they often generate false signals due to leaks in their systems.
  • To resolve this, researchers created a new reporter system that relies on combining gene fragments regulated by ribozymes to control the production of methyl halide gases.
  • This method demonstrated effective regulation, resulting in a significant increase in methyl halide detection during conjugation in soil, suggesting its potential for monitoring gene transfer and improving environmental biocontainment efforts.

Article Abstract

Enzymes that produce volatile metabolites can be coded into genetic circuits to report nondisruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halide gases. We split the gene into two nonfunctional fragments and attached these to a pair of splicing ribozyme fragments. While the individual -ribozyme fragments did not produce methyl halides when transcribed alone in , coexpression resulted in a spliced transcript that translated the MHT reporter. When cells containing one -ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second -ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

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Source
http://dx.doi.org/10.1021/acssynbio.4c00264DOI Listing

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