CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli

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 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 E. 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

Biofabrication with microbial cellulose: from bioadaptive designs to living materials

“…The combination of optimized culture media formulations and waste streams can reduce, to some degree, such risks, but a dedicated nutrient optimization should be ideally applied in high-throughput biofactory platforms. 232,233…”

“…The combination of optimized culture media formulations and waste streams can reduce, to some degree, such risks, but a dedicated nutrient optimization should be ideally applied in high-throughput biofactory platforms. 232,233 Taking advantage of the advancements in BNC fermentation and post-processing, another bioeconomy opportunity for BNC 3D biofabrication is that it can be integrated within preestablished industrial operations. For instance, acetic acid bacteria have been used to produce vinegar and kombucha via oxidizing ethanol to acetic acid, wherein BNC is a natural by-product of these industries; however, if the bioprocessing strategies could be adapted, such as enhancing the surface area of available oxygen upon BNC growth, the techniques discussed previously in Sections 3 and 4 could be implemented to realize BNC 3D biofabrication along with commercial vinegar or kombucha products.…”

Measurement Techniques to Resolve and Control Population Dynamics of Mixed-Culture Processes

“…Main challenges arise from the fact that the relationship between fluorescence and biomass concentration is not constant but varies depending on how the fluorescence tag is integrated genetically (e.g., types of promotor, free or fused protein construct) and the growth phase [104]. The latter can be explained by a mismatch between fluorescence protein production rate and growth rate.…”

Tunable population dynamics in a synthetic filamentous co-culture

“…High-throughput analysis has been attained using diverse approaches. For example, red and green uorescence monitoring in recombinantly labeled Escherichia coli co-cultures for biosensing of monoaromatics was described by Noonan et al [24]. Scattered light strain-speci c spectra have been evaluated as well.…”