CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli. Issue 1 (3rd September 2020)

• Record Type: Journal Article
• Authors: Noonan, Avery J C; Qiu, Yilin; Ho, Joe C H; Ocampo, Jewel; Vreugdenhil, K A; Marr, R Alexander; Zhao, Zhiying; Yoshikuni, Yasuo; Hallam, Steven J
• Is Part Of: Synthetic biology. Volume 5:Issue 1(2020)
• Publication Date: 2020-09-03
• Subjects: CRAGE -- biosensors -- microbial consortia -- co-culture dynamics -- functional screening; Synthetic biology -- Periodicals; 660.6
• DOI: 10.1093/synbio/ysaa015
• Languages: English
• ISSNs: 2397-7000

Abstract

Abstract: Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics in high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here, we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR -green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct Escherichia coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600 ) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications.