Table 1.
Concentrations of amino acids used in the competition study.
Figure 1.
Intracellular metabolite concentration ranges during batch overgrow CHO-S culture.
a) Glucose concentration measured by the Amplex Red ® glucose oxidase assay (gray circles) and glutamine concentration measured by a coupled glutaminase, glutamate dehydrogenase assay with correction for glutamate concentration (black squares) over the course of the batch overgrow culture. Error bars represent one standard deviation of the mean (n = 4 flasks with three enzymatic assay samples per flask) b) A closer view of the concentration of metabolites on days 4 to 8 (exponential to early stationary phase).
Figure 2.
Diagrammatic representation of FRET sensor function.
a) The glucose biosensor has maximum FRET efficiency in the absence of ligand. The binding of glucose causes a twist in the alignment of the fluorophores which causes the FRET ratio to decrease. b) The glutamine biosensor has maximum FRET efficiency in the presence of ligand which causes the binding domain to hinge closed, bringing the fluorophores into closer contact.
Figure 3.
In vitro titration data in comparison with published results.
a) Glucose biosensor measurements from Deuschle et al (2005) compared with titrations using purified protein extracted and measured as per the Materials and Methods section (n = 3 independent protein preparations, with 3 FRET ratio measurements per ligand concentration). b) Glutamine biosensor measurements from Yang et al (2010) compared with titrations titrations using purified protein extracted and measured as per the Materials and Methods section (n = 3 independent protein preparations, with 3 FRET ratio measurements per ligand concentration, error bars represent one standard deviation of the mean). Inset depicts a zoomed in view of the lower concentrations of glutamine.
Figure 4.
A comparison of the FRET measurements obtained from stable CHO-S cell lines constitutively expressing the biosensors and the corresponding measurements of intracellular concentrations from the same samples. a) Glucose biosensor measurements. Intracellular concentration as measured by the Amplex Red ® assay (gray circles) and corresponding FRET measurements (black squares). b) FRET versus concentration calibration curve for the glucose biosensor. The line represents the linear regression best fit for the data points. c) Glutamine biosensor measurements. Intracellular concentration as measured by the coupled glutaminase glutamate dehydrogenase assay (gray circles) and corresponding FRET measurements (black squares). d) FRET versus concentration calibration curve for the glutamine biosensor. The line represents the linear regression best fit for the data points. In all cases, n = 2 biological repeats with three measurements per cell line. Error bars represent one standard deviation of the mean.
Figure 5.
Assessment of the competition of other amino acids for the glutamine binding protein in the glutamine biosensor.
The ability of the purified biosensor protein to accurately report the concentration of glutamine in the presence of other amino acids was measured using the maximum intracellular concentration of amino acid encountered during cell culture and 1 mM glutamine. Results are plotted as the change in FRET ratio between each condition and the FRET ratio of the purified biosensor with no ligand present. n = 3. Error bars represent one standard deviation of the mean.
Figure 6.
Fed batch culture of CHO-S cells and corresponding FRET measurements.
a) Glucose fed-batch monitoring. Comparison of cells fed with glucose on day 6 (gray circles) and those given an equal volume of pure water (black squares). Arrow indicates addition of bolus feed to bring glucose concentration to 36 mM. b) Glutamine fed-batch monitoring. Comparison of cells fed with glutamine on day 6 (gray circles) and those given an equal volume of pure water (black squares). Arrow indicates addition of bolus feed to bring glutamine concentration to 4 mM. n = 3. Error bars represent one standard deviation of the mean.