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 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 P_emrR-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.

Keywords: CRAGE; biosensors; co-culture dynamics; functional screening; microbial consortia.

Figure 1.

CRAGE-enabled engineering of E. coli EPI300 reporter strains. (A) The emrR transcriptional regulator enables monoaromatic-dependent GFP expression following transformation with the pSB1C3-P_emrR-GFP biosensor plasmid. Four constitutive RFP expression cassettes, with a range of promoter strengths, were integrated into the chromosome as a method of labeling the reporter strain. (B) Chromosomal integration was performed using the CRAGE system, requiring landing pad transposition into the EPI300 chromosome. This enables high-efficiency Cre-lox-recombination into the CRAGE landing pad site. The location of integration was determined to be the yfaA gene. (C) RFP fluorescence/OD₆₀₀ (Normalized RFP) was calculated for the J23100, J23106, J23113 and J23114 strains after 24 h of incubation. Columns represent averages of two replicates. (D) OD₆₀₀ of CRAGE-engineered strains (J23100 and J23106) and EPI300 were monitored over 24 h (Time). OD₆₀₀ values represent averages of three replicates.

Figure 2.

Biosensor strain growth characteristics and response in monoculture. (A) In J23100 biosensor strain, with chromosomally integrated constitutively expressed RFP constructs, there is a non-linear relationship between RFUs (RFP fluorescence) and OD₆₀₀ (Optical density). Values represent single samples in 384-well format at various vanillin induction concentrations. (B) OD₆₀₀ (Optical density) values were measured hourly for 24 h (Time), at 7 vanillin concentration ranging from 0 to 640 µM, to observe the impact of vanillin induction on growth rate of biosensor strains. Biosensor response was observed in RFP-labeled strains to determine the impact of co-expression of vanillin-induced GFP and constitutively expressed RFP. OD₆₀₀ values represent single samples in 384-well format at various vanillin induction concentrations. (C) The response of the P_emrR-GFP biosensor (Normalized GFP) to vanillin induction at 7 concentrations (Vanillin) is shown at 14 h after inoculation of the J23100 strain. Boxes were set using three single sample replicates in 384-well format. (D) RFP fluorescence in RFUs in the J23100 strain over 24 h (Time), at 7 vanillin induction concentrations. RFP fluorescence values represent single samples in 384-well format at various vanillin induction concentrations.

Figure 3.

Characterizing the relationship between RFP fluorescence and OD₆₀₀ over the E. coli growth cycle. (A) The ratio of RFP RFUs/OD₆₀₀ (Normalized RFP) plotted over 24 h (Time) in strain J23100, at 7 vanillin induction levels, shows the non-linearity of RFP fluorescence/OD₆₀₀. Values represent single samples in 384-well format. (B) A linear relationship between RFU/OD₆₀₀ (Normalized RFP) and time (Time) can be observed during the deceleration phase of the E. coli growth cycle. This requires defining T₀ of this relationship as the entry into deceleration phase. Points represent single samples in 384-well format at 7 induction concentrations. The linear trend is represented by Equation 3. (C) The minimum (t = 8) and maximum (t = 12) in the double-derivative of the growth curve (δ²OD/δT²) of J23100 indicate times (Time) of the largest positive and negative rates of change of growth rate. Values represent averages of 21 samples at 7 induction concentrations. (D) The relative increase in RFP fluorescence (Relative δRFP/δT) over time shows a maximum (t = 12) at the time (Time) of entry into deceleration phase. Values represent averages of 21 samples at 7 induction concentrations.

Figure 4.

Monitoring bulk co-culture behavior. (A) Co-culture-level growth characteristic (Optical density) did not differ significantly between vanillin concentration over time (Time). (B) At a single timepoint (T₁₅), neither inoculation ratio (Biosensor Inoculation %) or vanillin induction concentration had a significant impact on measured OD₆₀₀ values (Optical density). (C and D) However, variation in the initial inoculation density of the biosensor strain (Biosensor Inoculation %), as well as the concentration of vanillin, both had a significant impact on fluorescence of RFP and GFP (GFP/RFP). All values represent averages of three replicates.

Figure 5.

Calculating proportions of J23106 biosensor strain in co-culture using RFUs and predicting biosensor response through GFP normalization to calculated OD₆₀₀. (A) At a vanillin concentration of 0 µM, OD₆₀₀ values (Optical density) remained relatively consistent in co-culture, regardless of biosensor inoculation ratios. (B) However, predicted OD₆₀₀ of the biosensor strain (Predicted OD₆₀₀) were proportional to inoculation densities. (C) At a biosensor strain inoculation density of 75%, co-culture level OD₆₀₀ values (Optical density) remained consistent, (D) whereas predicted OD₆₀₀ values decrease with increasing vanillin concentration (0 µM, 320 µM and 640 µM). All values represent averages of three replicates. (E) GFP fluorescence was normalized to calculated biosensor-specific OD₆₀₀ (Predicted GFP/OD₆₀₀), at increasing inoculation densities (Biosensor inoculation %) and vanillin concentrations. Values represent averages of three replicates. (F) At biosensor inoculation ratios of 50% or greater, the increase in relative GFP expression (Predicted GFP/OD₆₀₀) in response to vanillin (Vanillin) was consistent across ratios. There was minimal variation between calculated normalized GFP fluorescence at 0 µM Vanillin. Points represent single samples.

Figure 6.

Determining population dynamics using flow cytometry. Proportions of strains in samples collected 15 h after inoculation are shown. At a vanillin concentration of 0 µM, relative abundances were proportional to their inoculation ratios of 90% (A), 50% (B) and 10% (C) J23100 biosensor strain (Y-axis = Side scatter/X-axis = GFP fluorescence). Plots represent events from single samples. Increased vanillin concentration from 0 µM to 320 µM led to a relative increase in EPI300-pCC1 abundance at inoculation ratios of 90% (D), 50% (E) and 10% (E) biosensor strain (Y-axis = Count/X-axis = GFP fluorescence). Histograms represent counts of two samples at either 0 µM (gray) or 320 µM (blue) vanillin.

Figure 7.

Model based prediction of OD₆₀₀ values in co-culture compared to measured cell counts. Measured and predicted OD₆₀₀ values (Optical density) at T₁₅ are compared at two induction concentrations (0 µM and 320 µM vanillin) and three inoculation proportions (10%, 50% and 90% biosensor strain). Similar trends were observed in both the J23100 (A) and J23106 (B) biosensor strains. Each bar represents either a single flow cytometry analysis or a single predicted OD₆₀₀ value of the biosensor strain.