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Although the genetic makeup of an organism plays a major role in determining the phenotype, there are also several environmental factors, such as temperature, oxygen availability, presence of mutagens, that can alter an organism’s phenotype.
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Influence of Environmental Conditions on the Escape Rates of Biocontained Genetically Engineered Microbes.

Anna M Hartig1, Wentao Dai1, Ke Zhang1

  • 1Department of Energy, Environmental, and Chemical Engineering, Washington University in St. Louis, St. Louis, Missouri 63130, United States.

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Genetically engineered microbes (GEMs) require robust biocontainment. Kill switch effectiveness dramatically decreases in natural environments due to factors like pH and nutrient levels, increasing GEM escape rates.

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CRISPR-Cas9biocontainmentbiosafetyescape ratesgenetically engineered microbeskill switchrisk assessment

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Area of Science:

  • Environmental microbiology
  • Synthetic biology
  • Biotechnology

Background:

  • Genetically engineered microbes (GEMs) offer significant potential but pose environmental risks.
  • Biocontainment strategies, such as kill switches, are crucial for controlling GEM proliferation.
  • Current kill switch assessments often occur in lab conditions, not reflecting real-world environmental challenges.

Purpose of the Study:

  • To evaluate the environmental effectiveness of a CRISPR-based kill switch in *Escherichia coli* GEMs.
  • To investigate the impact of environmental conditions on kill switch performance and GEM escape rates.
  • To identify factors contributing to kill switch failure in natural settings.

Main Methods:

  • Deployment of an *Escherichia coli* GEM with a CRISPR-based kill switch in natural surface waters and laboratory media.
  • Assessment of kill switch escape rates under varying environmental conditions.
  • Analysis of environmental factors (pH, nutrient availability) influencing kill switch trigger efficacy and microbial viability.

Main Results:

  • The escape rate of the GEM with a CRISPR-based kill switch increased by 3-4 orders of magnitude in natural surface waters compared to laboratory media.
  • Environmental factors, including pH and nutrient levels, significantly impacted kill switch function.
  • Altered chemical speciation of the anhydrotetracycline (aTc) trigger and limited nutrients reduced kill switch efficacy.

Conclusions:

  • Kill switch effectiveness is highly dependent on environmental conditions, often underestimating failure rates in laboratory settings.
  • Environmental factors must be considered during the design phase to ensure effective biocontainment of GEMs.
  • Developing robust GEM biocontainment requires a thorough understanding of the target environment.