Flow cytometric analysis of E. coli on agar plates: implications for recombinant protein production

Abstract

Recombinant protein production in bacterial hosts is a commercially important process in the pharmaceutical industry. Optimisation of such processes is of critical importance for process productivity and reproducibility. Here, flow cytometry methods were developed to assess characteristics of bacteria during two process steps that are infrequently studied: agar plate culture and liquid culture set-up. During storage on agar plates, three discrete populations of varying green fluorescence intensity were observed along with a progressive shift of cells from the high green fluorescence population to an intermediate green fluorescence population, observed to be due formation of amyloid inclusion bodies. The dynamics of cellular fluorescence and scatter properties upon setup of liquid cultures were also assessed. These methods have the potential to improve the development of fermentation set-up, a currently little-understood area.

Heterogeneity in Pure Microbial Systems: Experimental Measurements and Modeling

“…The effect of cell individuality when using bacteria for obtaining useful products has been emphasized recently in several publications ( Li and You, 2013 ; Wyre and Overton, 2014a , b ; Chen et al, 2015 ). Nevertheless, the true impact of microbial population heterogeneity on bioprocesses remains unknown ( Delvigne and Goffin, 2014 ) and therefore it is not systematically considered in design.…”

Optimisation of recombinant TNFα production in Escherichia coli using GFP fusions and flow cytometry

“…Induced cultures showed a similar pattern up to 10 h growth, although at 28 h cultures expressing both GFP and TNFα-GFP comprised multiple populations with different green fluorescence, likely reflecting subpopulations with different RP accumulation and folding state ( Wyre and Overton, 2014a ). A maximum of 7%–10% of cells were GFP − (around 2 × 10 2 fluorescence units, representing E. coli autofluorescence and the same as untransformed cells, Supplementary Figure S2 ) indicating that plasmid selection was sufficient to prevent formation of a significant plasmid-free population.…”

“…Translational fusion of gfp at the C-terminal of a recombinant gene of interest permits measurement of fluorescence which correlates to the quantity and folding state of the recombinant protein; if the recombinant protein folds correctly then GFP also tends to fold correctly and becomes fluorescent, whereas recombinant protein misfolding leads to GFP misfolding and low fluorescence ( Waldo et al, 1999 ; Vera et al, 2007 ). We have previously used FCM to optimise production of a model recombinant protein ( E. coli CheY, which readily misfolds when generated at high levels) fused to GFP in both lab-scale ( Sevastsyanovich et al, 2009 ) and bioreactor cultures ( Wyre and Overton, 2014b ), as well as at other stages of the RPP process ( Wyre and Overton, 2014a ).…”

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

“…Expression systems using two or more plasmids may present high instability, thus enabling their use only as ‘‘transient’’ expression systems (Al-Allaf et al, 2013 ). During cell growth in high-cell-density cultures, overexpression of recombinant proteins often results in (i) cell aggregation and (ii) bacteria becoming viable but non-culturable, a phenotype which means the loss of the ability to form colonies on agar plates (Jeong & Lee, 2003 ; Wyre & Overton, 2014a ; Fernández-Castané et al, 2017 ). Flow cytometry (FCM) has been extensively used as a rapid and high-throughput analytical technique to determine the viability and physiology of cells and to detect heterogeneities in cultures (Hewitt et al, 1999 ; Wyre and Overton, 2014b ).…”