Assessing the growth kinetics and stoichiometry of Escherichia coli at the single‐cell level

Abstract

Microfluidic cultivation and single-cell analysis are inherent parts of modern microbial biotechnology and microbiology. However, implementing biochemical engineering principles based on the kinetics and stoichiometry of growth in microscopic spaces remained unattained. We here present a novel integrated framework that utilizes distinct microfluidic cultivation technologies and single-cell analytics to make the fundamental math of process-oriented biochemical engineering applicable at the single-cell level. A combination of noninvasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and picoliter batch reactors, enabled the quantification of all physiological parameters relevant to approximate a material balance in microfluidic reaction environments.We determined state variables (biomass concentration based on single-cell dry weight and mass density), biomass synthesis rates, and substrate affinities of cells grown in microfluidic environments. Based on this data, we mathematically derived the specific kinetics of substrate uptake and growth stoichiometry in glucose-grown Escherichia coli with single-cell resolution. This framework may initiate microscale material balancing beyond the averaged values obtained from populations as a basis for integrating heterogeneous kinetic and stoichiometric single-cell data into generalized bioprocess models and descriptions.

Transport‐controlled growth decoupling for self‐induced protein expression with a glycerol‐repressible genetic circuit

“…A K S of approx. 0.5 mg L −1 has been reported for wild‐type E. coli MG1655 (Smaluch et al, 2023), while the estimated K S of strain WGM is 10 mg L −1 (Krausch et al, 2023). This 20‐fold decreased affinity may result in a growth rate controlled by the glucose concentration at the end of the culture, which can be detected due to the relatively high cell density.…”

“…It was shown that the cAMP‐Crp complex pool is a function of the glucose uptake rate (You et al, 2013) and probably sensitive to the carbon uptake rate (Okano et al, 2020). The affinity for glucose is heterogeneous in cultures of E. coli (Smaluch et al, 2023), and thus q gluc can also vary from cell‐to cell. Overall fitness is also heterogeneous in bacterial cultures (Henrion et al, 2023).…”

“…The affinity for glucose is heterogeneous in cultures of E. coli (Smaluch et al, 2023), and thus q gluc can also vary from cell-to cell.…”

Adaptive control of the E. coli-specific growth rate in fed-batch cultivation based on oxygen uptake rate

“…The specific growth rate values for the SGR control system were obtained in conjunction with the evaluation of the biomass concentration. The following equation was used to calculate the SGR for the time interval [42] : where μ is the specific growth rate.…”

“…The values proposed in Table 3 , as well as the initial values ( =0.5 g kg −1 , =5 g kg −1 , =8 kg), were taken from the research works of [17] , [28] , [42] . The controller parameter was manually recalibrated in the cultivation process data of the BL21 (DE3) pET21-IFN-alfa-5 strain.…”