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Adaptive Laboratory Evolution of Probiotics toward Oxidative Stress Using a Microfluidic-Based Platform.

Ann V Nguyen1, Mohammad Yaghoobi1, Shiying Zhang2

  • 1Department of Food Science, College of Agricultural and Life Sciences, Cornell University, Stocking Hall, Ithaca, NY, 14853, USA.

Small (Weinheim an Der Bergstrasse, Germany)
|January 22, 2024
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Summary

Adaptive laboratory evolution using a novel microfluidic chip enhanced Lacticaseibacillus rhamnosus GG

Keywords:
adaptive laboratory evolutionadaptive strainslab‐on‐a‐chipoxidative stressprobiotics

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Area of Science:

  • Microbiology
  • Biotechnology
  • Synthetic Biology

Background:

  • Adaptive laboratory evolution (ALE) enhances bacterial resistance to oxidative stress.
  • Microfluidic platforms offer efficient and economical alternatives to traditional batch cultures for ALE.
  • Existing microfluidic ALE systems face challenges with cell passaging and spatial control.

Purpose of the Study:

  • To develop and evaluate a novel microfluidic Evolution on a Chip (EVoc) system for ALE.
  • To enhance the oxidative stress tolerance of Lacticaseibacillus rhamnosus GG using the EVoc platform.
  • To investigate the adaptive mechanisms and genetic changes in L. rhamnosus GG under prolonged H2O2 exposure.

Main Methods:

  • Utilized a microfluidic EVoc design with a progressive H2O2 gradient.
  • Performed prolonged ALE (up to 72 hours) on L. rhamnosus GG.
  • Analyzed adaptive strain morphology, gene expression, and performed whole-genome sequencing.

Main Results:

  • Successfully evolved adaptive strains of L. rhamnosus GG exhibiting increased H2O2 tolerance.
  • Demonstrated progressive growth enhancement in increasing H2O2 concentrations (1-3 mM) after ALE.
  • Observed distinct morphological and gene expression profiles in adapted strains compared to wild-type.
  • Identified a single nucleotide mutation in the omega-amidase gene in adapted strains.

Conclusions:

  • The microfluidic EVoc platform is effective for ALE, enhancing bacterial oxidative stress resistance.
  • ALE significantly improves L. rhamnosus GG's ability to tolerate and grow in the presence of hydrogen peroxide.
  • Genetic adaptation, including a mutation in omega-amidase, underlies the enhanced stress tolerance.