Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

Abstract

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-␤-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by MichaelisMenten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

Linking environmental heterogeneity and reproductive success at single-cell resolution

“…GFP dilution is a quantitative measure for reproductive success A previously formulated mathematical model of GFP expression in bacteria (Leveau and Lindow, 2001b) predicts that in the absence of de novo synthesis, GFP dilutes from dividing cells at a rate equal to growth rate m. We verified this prediction here using Eh299RHJBA28 (pCPP39), which accumulates stable GFP in the presence of the synthetic inducer IPTG. Upon transfer of IPTG-induced GFPloaded cells to broth that lacked IPTG, GFP fluorescence declined exponentially and at a rate that was not significantly different from m (Figure 1).…”

“…Another plus of the reproductive success bioreporter is that it offers microbial ecologists low-ambiguity output. Most GFP bioreporters are promoter-based, and although promoters can be quite specific in response to the environmental variable under investigation, their activity can be modulated in unpredictable ways by other input from the environment (Leveau and Lindow, 2001b). Such ambiguity makes promoter-based bioreporters susceptible to misinterpretation, particularly in the absence of proper controls (Leveau and Lindow, 2001b).…”

“…Most GFP bioreporters are promoter-based, and although promoters can be quite specific in response to the environmental variable under investigation, their activity can be modulated in unpredictable ways by other input from the environment (Leveau and Lindow, 2001b). Such ambiguity makes promoter-based bioreporters susceptible to misinterpretation, particularly in the absence of proper controls (Leveau and Lindow, 2001b). The reproductive-success bioreporter is promoter-independent in that it is solely based on dilution of previously synthesized GFP from the cell by division, hence with the minimal likelihood of misinterpretation of GFP output.…”

“…Bacterial strains and culture conditions Erwinia herbicola 299RHJBA28 (pCPP39) (Eh299RHJBA28 (pCPP39)) (Leveau and Lindow, 2001b) carries a chromosomal mini-Tn5-Km transposon insertion that expresses stable GFP from a LacI q -repressible P A1/O4/O3 promoter fusion to gfpmut3. The transposon confers resistance to kanamycin.…”

Synthetic Biology: opportunities for Chilean bioindustry and education

“…The use of in vivo fl uorescent markers, in combination with mathematical descriptions, has been critical to deriving estimates of underlying mechanisms, such as transcription rates (De Jong et al, 2010;Leveau et al, 2001;Kelly et al, 2009;Munsky et al, 2012), parameters of induction/repression, and translation effi ciency . In most cases, these estimates are indirect measurements in arbitrary units because the fl uorescence level is used as a proxy for underlying processes that are not accessible to measurement.…”