PMC

Liquid distribution measurement in shake flasks using an optical fluorescence technique. a shows a schematic diagram of the liquid distribution measurement setup for 250-mL shake flask. b shows a top view of the optical fiber arrangement at the side of the shake flask wall. c, d and e show a side view, close-up view and 45^o distant view of the optical fiber arrangement to the shake flask, respectively. 1) Orbital shaker. 2) Hall sensor. 3) Magnet for triggering. 4) Light-emitting diode (LED) supplying excitation blue light (465 nm). 5) Optical fiber channeling the excitation blue light to the shake flask. 6) 45^o angle distance of the optical fiber arrangement (seen from top view). 7) 250-mL shake flask clamped onto the orbital shaker’s table. 8) Enlarged view of the 14 different measurement positions to the side of the shake flask wall. Fourteen pairs of optical fibers were arranged vertically at these specified different heights from the base of the shake flask. 9) Optical fiber channeling the emission light (515 nm) from the shake flask to the photomultiplier tube (PMT). 10) Enlarged view of the bundle of 14 optical fibers. 11) Enlarged view of the PMT’s light sensitive area attached with a bundle of 14 optical fibers. 12) Data acquisition module (analog-to-digital converter). 13) Computer. 14) Brass holder for optical fibers. 15) Transparent core of optical fiber. 16) Shake flask wall. 17) Rotating fluorescent aqueous solution

Fermentation of strain harboring CRISPRi system to repress by-product pathways. (A) Biomass (OD₆₀₀) of the CRISPRi strain in the shake flask. (B) Glucose consumption of CRISPRi strain in the shake flask. (C) Glycerol production by CRISPRi strain in the shake flask. (D) 3-Hydroxypropionic acid production by CRISPRi strain in the shake flask. (E) Lactic acid production by CRISPRi strain in the shake flask. (F) Acetic acid production by CRISPRi strain in the shake flask. (G) 1,3-Propanediol production of CRISPRi strain in the shake flask. (H) 2,3-Butanediol production by CRISPRi strain in the shake flask. (I) Metabolites of CRISPRi strain KP-GB2 in the shake flask. (J) Metabolites of CRISPRi strain KP-GB2 in fed-batch culture. KP-dCas9, the recombinant K. pneumoniae strain with dCas9 integrated in genome; KP-15A, the recombinant K. pneumoniae strain harboring empty vector ptac-15A; KP-GS, the recombinant K. pneumoniae with dCas9 integrated in genome, and harboring vector ptac-GS co-expressing non-target sgRNA, gpd1 and gpp2; KP-GG1, recombinant K. pneumoniae with dCas9 integrated in genome of and ptac-GG1 plasmid co-expressing gpd1, gpp2, and gapA-targeting sgRNA; KP-GB2, the recombinant K. pneumoniae with dCas9 integrated in genome and vector ptac-GB2 co-expressing gpd1, gpp2, and budA-targeting sgRNA; KP-GGB, the recombinant K. pneumoniae with dCas9 integrated in genome and plasmid ptac-GGB co-expressing gpd1, gpp2, and sgRNAs targeting gapA and budA. 3-HP, 3-hydroxypropionic acid; LA, lactic acid; AA, acetic acid; 1,3-PDO, 1,3-propanediol; and 2,3-BDO, 2,3-butanediol.

Impact of glutamate concentration on cell growth and metabolism in Datachip and shake flask platforms. Changes in (A) cell growth in DataChip platform, (B) cell growth in shake flask platform, (C) glucose metabolism DataChip platform, (D) glucose metabolism shake flask platform, (E) lactate production DataChip platform, (F) lactate production shake flask platform, (G) glutamate concentration DataChip platform and, (H) glutamate concentration shake flask platform. Cell growth in DataChip platform was determined using cell viability assay. Cell growth in shake flask platform was determined by manual cell counting using trypan blue, according to the standard operating protocol provided by J&J, Janssen Biotech Metabolite analysis on cell-conditioned media collected from the chip samples and shake flask samples were performed using an YSI analyzer. DataChip data represents the mean ± SE of an experiment with 96 replicate microcultures and pooled spent media from the 96 microcultures for metabolite analysis. Shake flask data represents the mean ± standard deviation, where, n=2 independent experiments.

A histogram of metabolite residuals, x−x, reveals the error structure between samples and shake flasks for wild-type aerobic intracellular metabolites. The residuals do not display a large flask bias relative to the overall sample variability. Residuals from the other data classes behaved similarly. Thus we adopt the equal-means hypothesis among shake flasks and samples from each shake flask were treated as independent repetitions.