Biocontainment strategies, such as kill switches, have been developed to avoid the unintended proliferation of genetically engineered microbes (GEMs) intended for open-release environmental applications. However, the presence of GEM DNA after successful biocontainment presents new environmental risks and challenges for monitoring. In this study, we investigated whether biocontainment using a CRISPR-Cas9 kill switch, which causes double-strand breaks in target genes essential for GEM growth, could resolve this challenge in a model Escherichia coli GEM. Surprisingly, the escape rates of the GEM as determined by CRISPR-targeted gene abundances were as high as 10-1.6 to 10-1.0 in LB media, despite the escape rates measured by colony forming units (cfu) being only 10-6.2 under the same condition. This discrepancy suggested that the CRISPR-Cas9 kill switch prevents colony growth while still leaving a large fraction of target genes intact for detection by molecular methods. Within 1 h after biocontainment, these target genes remained predominantly inside an intact cell membrane and were resistant to degradation by DNase, though degradation was observed in river water over multiple days. Overall, a detailed understanding of the impact of the biocontainment mechanism on both the GEM and its DNA is needed to minimize unintended environmental risks.
Gas-particle partitioning is critical for the evolution of secondary organic aerosols (SOA) in the atmosphere. SOA particles evaporate more slowly than expected at nearly size-independent rates, but the underlying mechanism remains controversial. Here, we apply kinetic multilayer modeling to simulate evaporation of α-pinene SOA, demonstrating that surface crust formation, emerging from accumulation of low-volatility compounds at the particle surface, leads to slow evaporation and reduced size dependence of the evaporation rate. While evaporation induced by decomposition of oligomers would naturally lead to size-independent evaporation rates, we observe and simulate nearly size-independent slow evaporation of polyethylene glycol mixture particles containing polymeric species that do not decompose, confirming the relevance of composition-dependent diffusivity for size-independent, slow evaporation. Slow evaporation of limonene SOA was also observed in environmental chamber experiments, and model simulations demonstrate strong surface crust formation with bulk diffusivity being depressed by up to 5 orders of magnitude compared to the inner bulk. We present experimental evidence using a surface-based mass spectrometry technique that shows that the particle surface becomes enriched in high molecular weight compounds upon evaporation of monomers. Our findings imply that viscous surface crusts may also limit the growth and chemical transformation of SOA particles, influencing their impacts on air quality and climate.
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