Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Multiple Genes in a Single Host: Cost-Effective Production of Bacterial Laccase (cotA), Pectate Lyase (pel), and Endoxylanase (xyl) by Simultaneous Expression and Cloning in Single Vector in E. coli.

This study attempted to reduce the enzyme production cost for exploiting lignocellulosic materials by expression of multiple genes in a single host. Genes for bacterial laccase (CotA), pectate lyase (Pel) and endoxylanase (Xyl), which hold significance in lignocellulose degradation, were cloned in pETDuet-1 vector containing two independent cloning sites (MCS). CotA and xyl genes were cloned in MCS1 and MCS 2, respectively. Pel gene was cloned by inserting complete cassette (T7 promoter, ribosome binding site, pel gene, His tag and complete gene ORF) preceded by cotA open reading frame in the MCS1. IPTG induction of CPXpDuet-1 construct in E. coli BL21(DE3) resulted in expression of all three heterologous proteins of ~65 kDa (CotA), ~45 kDa (Pel) and ~25 kDa (Xyl), confirmed by SDS-PAGE and western blotting. Significant portions of the enzymes were also found in culture supernatant (~16, ~720 and ~370 IU/ml activities of CotA, Pel and Xyl, respectively). Culture media optimization resulted in 2, 3 and 7 fold increased secretion of recombinant CotA, Pel and Xyl, respectively. Bioreactor level optimization of the recombinant cocktail expression resulted in production of 19 g/L dry cell biomass at OD600nm 74 from 1 L induced culture after 15 h of cultivation, from which 9, 627 and 1090 IU/ml secretory enzyme activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to increase the saccharification of orange peel in comparison to the xylanase alone. Thus, simultaneous expression as well as extra cellular secretion of these enzymes as cocktail can reduce the enzyme production cost which increases their applicability specially for exploiting lignocellulosic materials for their conversion to value added products like alcohol and animal feed

Comparison of the hydrolyzing efficiency of CPX enzyme cocktail and Xyl alone by sugar release estimation at uniform time intervals.

<p>Comparison of the hydrolyzing efficiency of CPX enzyme cocktail and Xyl alone by sugar release estimation at uniform time intervals.</p

Bioreactor level optimization of the recombinant enzyme cocktail production.

<p>(a) Change in DO concentration with respect to change in RPM; (b) Increase in dry cell weight (DCW) with respect to change in OD_600nm, and DO concentration; (c) Increase in the activities of CotA, Pel and Xyl with respect to DCW; and (d) Change in concentration of glucose and glycerol with respect to DCW and OD_600nm.</p

Vector map of CPXpETDuet-1 (Plotted using SimVector4.0 software).

<p>Vector map of CPXpETDuet-1 (Plotted using SimVector4.0 software).</p

Western blotting analysis of the expression of recombinant cocktail in <i>E</i>. <i>coli</i> BL21(DE3).

<p>Lane 1: Protein MW marker; Lane 2: Supernatant of induced culture; Lane 3: Cell biomass of induced culture; Lane 4–5: Western blot of CS and cell biomass showing no signal in the supernatant which might be due to removal of N-terminal signal sequence along with His tag during secretion.</p

Primer sequences used for amplification of CotA, Pel and Xyl genes.

<p>Primer sequences used for amplification of CotA, Pel and Xyl genes.</p

Comparison of the Activities of Pel, CotA and Xyl between cocktail and individual gene expression.

<p>Comparison of the Activities of Pel, CotA and Xyl between cocktail and individual gene expression.</p

Activity of CotA (a), Xyl (b) and Pel (c) in CS, PF and CF when expressed in <i>E</i>. <i>coli</i> BL21(DE3), <i>E</i>. <i>coli</i> BL21(DE3) pTUM4 and <i>E</i>. <i>coli</i> BL21(DE3) Arctic.

<p>Activity of CotA (a), Xyl (b) and Pel (c) in CS, PF and CF when expressed in <i>E</i>. <i>coli</i> BL21(DE3), <i>E</i>. <i>coli</i> BL21(DE3) pTUM4 and <i>E</i>. <i>coli</i> BL21(DE3) Arctic.</p

Comparison of CPX expression when cultivated in LB, wheat bran and terrific broth.

<p>Significant amount of pectate lyase and endoxylanase was observed in CS. Lane 1: Protein MW marker; Lane 2: LB uninduced control culture; Lane 3–4: LB induced culture cell biomass and CS; Lane 5–7: LB+wheat bran induced culture CF pellet, CF sup and CS; Lane 8–10: terrific broth induced culture CF pellet, CF sup and CS.</p