Liquid Soil Models for Measuring Survival and Performance of Engineered Pseudomonas Putida

Biosensors, bioremediation, and pathogen control using synthetic organisms are among the solutions for soil contamination that have been developed yet, we lack sufficient understanding of how to make the engineered bacteria survive and perform optimally within the complex soil environment. The soil microbial community (SMC), is an essential part of understanding the survival and persistence of genetically engineered microbes (GEM). However, when GEMs encounter the soil, their population is reduced and surpassed by natural organisms. To test the survival of our engineered organism Pseudomonas putida in the lab, we created a Soil Extracted Solubilized Organic Matter (SESOM) or sterile liquid soil extract. Over the course of 28 days, we employed 16S rRNA amplicon sequencing to follow changes in soil communities, and we used flow cytometry to examine the dynamic survival of P.putida expressing the tdTomato fluorescent marker in sterile and non-sterile SESOM. The taxonomy study revealed that the most prevalent bacterial classes in community soil are Alphaproteobacteria, Bacteroidia, Actinobacteria, and Gammaproteobacteria. The samples over the course of 28 days also revealed similarities between solid soil and non-sterile liquid soil extract (NS-SESOM). P. Putida growth curve in SESOM was found to be comparable to that of P. Putida growing in LB, although there is a population decline when P. Putida grows in NS-SESOM. These findings demonstrate that the results obtained in liquid soil extract mirror the solid soil samples. This system is a scalable and automated laboratory model that can forecast GEM survival and persistence, enabling its implementation in the soil.