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The genome of most prokaryotic organisms consists of double-stranded DNA organized into one circular chromosome in a region of cytoplasm called the nucleoid. The chromosome is tightly wound, or supercoiled, for efficient storage. Prokaryotes also contain other circular pieces of DNA called plasmids. These plasmids are smaller than the chromosome and often carry genes that confer adaptive functions, such as antibiotic resistance.
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Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach
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Engineering E. coli strains using antibiotic-resistance-gene-free plasmids.

Matthew B Amrofell1, Sunaina Rengarajan2, Steven T Vo1

  • 1Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.

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|December 12, 2023
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Summary

We developed a novel cloning method using antibiotic-resistance-gene-free plasmids (ARGFPs) and a dual selection strategy. This approach ensures efficient plasmid maintenance in engineered bacteria, reducing antibiotic resistance gene spread.

Keywords:
CP: BiotechnologyCP: Microbiologyantibiotic resistancecloningguthorizontal gene transferplasmidprobiotic

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Molecular Biology

Background:

  • Antibiotic resistance gene (ARGs) based cloning poses risks of horizontal gene transfer.
  • Engineered bacterial strains are crucial for probiotic applications.
  • Efficient and safe plasmid-based cloning methods are needed.

Purpose of the Study:

  • To develop a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning.
  • To establish a dual selection strategy for constructing and maintaining plasmids in engineered E. coli strains.
  • To demonstrate the utility of this method in common laboratory and probiotic chassis.

Main Methods:

  • Utilized a dual auxotrophic- and essential-gene-based selection strategy.
  • Constructed plasmids in engineered E. coli DH10B cloning strains.
  • Selected and maintained plasmids in E. coli Nissle 1917 and E. coli MG1655.

Main Results:

  • Achieved comparable cloning efficiency to traditional antibiotic-resistance-gene-based methods.
  • Demonstrated ease of transformation in double-knockout Nissle and MG1655 strains.
  • Confirmed long-term plasmid maintenance in engineered Nissle strains through repeated culturing and in vivo mouse gut studies.

Conclusions:

  • The developed ARGFP-based cloning pipeline is efficient and generalizable.
  • This method minimizes the risk of antibiotic resistance spread via horizontal gene transfer.
  • Engineered E. coli Nissle strains are suitable for long-term plasmid maintenance in various applications, including probiotic development.