The Effects of Intraspecies and Interspecies Competition on Genetic Device Construction and Performance

Abstract

Future genetic devices expressed in microbes need to be tested in complex environments containing multiple organisms to expand their usability. We sought to determine if genetic parts designed for monoculture are predictable when used in co-culture by testing constitutive Anderson promoters driving expression of chromoproteins from a plasmid. In Escherichia coli monoculture a high copy number origin of replication causes stochastic expression regardless of promoter strength, and high Anderson promoter strength leads to selection for inactivating mutations, resulting in inconsistent chromoprotein expression. Medium and low strength Anderson promoters function more predictably in E. coli monoculture but experience an increase in inactivating mutations when grown in co-culture over many generations with Pseudomonas aeruginosa. Expression from native promoters instead of Anderson promoters can lead to stable expression in a complex wastewater culture. Overall, we show intraspecies selection for inactivating mutations due to a competitive growth advantage for E. coli that do not express the genetic device compared to their peers that retain the functional device. We show additional interspecies selection against the functional device when E. coli is co-cultured with another organism. Together, these two selection pressures create a significant barrier to genetic device function in microbial communities that we overcome by utilize a native E. coli promoter. Future strategies for genetic device design in microorganisms that need to function in a complex microbial environment should focus on native promoters and/or strategies that give the microorganism carrying the device a selective or growth advantage. ImportanceFirst generation biotechnology focused on genetic devices designed for use in monoculture conditions. Next generation biotechnology devices will need to function in complex ecosystems with other organisms, so we sought to create conditions where the genetic device retained function when the organism carrying it is in co-culture with other organisms. We discovered that when the genetic device is a significant resource burden on the organism carrying the device mutations will be selected for due to intraspecies and interspecies selection pressures and the device will be rendered non-functional. Therefore, genetic device design for complex ecosystems in next generation biotechnology needs to balance functionality of the genetic device with the need to reduce resource burden on the organism carrying it.