Temporal variation of recombinant protein expression in Escherichia coli biofilms analysed at single-cell level

Abstract

Highlights Epifluorescence microscopy and image processing enable single-cell expression analysis. Escherichia coli biofilm heterogeneity increased during biofilm development. Fluorescence heterogeneity was correlated with spatial heterogeneity. 2 AbstractBioprocesses based on surface-associated microorganisms are emerging in environmental and industrial areas owing to the physiological specificities and heterogeneities of biofilm cells. This study describes a simple and accurate method for evaluating recombinant protein expression at a single-cell scale during Escherichia coli biofilm development. The model recombinant protein used was enhanced Green Fluorescent Protein (eGFP), as its intrinsic fluorescence allows the quantification of expression at both population and single-cell levels. Specific cell fluorescence intensity sharply increased during the first 4 days of biofilm cultivation and thereafter decreased abruptly to reach a low-level plateau until the end of the experiment. During biofilm development, the population became increasingly heterogeneous with regard to eGFP expression. Three distinct biofilm types were observed along the experimental time: one with a homogeneous population (days 3-5), the second with a moderately heterogeneous population (days 6-8) and the third with a strongly heterogeneous population (days 9-11). Observation of E. coli biofilms by confocal laser scanning microscopy demonstrated marked spatial heterogeneity, with the cells actively producing eGFP restricted to the top layer of the biofilm. The proposed methodology allows a fine analysis of the recombinant protein expression within E. coli biofilms, and it may be used for optimizing the processing conditions. Keywords: Biofilm; Escherichia coli; recombinant protein expression; fluorescence microscopy; single-cell scale; spatial heterogeneity Introduction 3The Gram-negative bacterium Escherichia coli is one of the most commonly used recombinant protein production hosts 1 due to its ability to grow rapidly and to high densities on inexpensive substrates, its well-known genetics and the availability of various systems for gene expression.2 Several bacteria such as E. coli naturally grow in a community attached to a substratum and not in liquid cultures. The biocatalytic potential of these bacterial communities, termed biofilms, can be attributed to their high cell density; the former feature is widely used for wastewater treatment 3 and also for the production of industrial chemicals such as ethanol, butanol and lactic acid. 4,5 Recombinant protein production in biofilms has been mostly studied in the context of waste biodegradation 6,7 ; however, this strategy could also be advantageous in other processes such as the biosynthesis of pharmaceutical intermediates 8 and catalysts for the food industry. In fact, using a recombinant Aspergillus niger strain, which contained a gene encoding the glucoamylase-GFP fusion protein, Talabardon and Yang 9 showed that higher amounts of GFP (Green Fluorescent Protein) and glucoamylase...

The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms

“…Then, the biofilm was resuspended and homogenized in 25 mL of 8.5 g L −1 NaCl solution for assessment of total and viable cells and for quantification of eGFP and plasmid content. The Live/Dead ® BacLight™ bacterial viability kit (Syto9/propidium iodide; Invitrogen Life Technologies, Alfagene, Carcavelos, Portugal) was used to quantify the viability of bacterial populations as a function of the membrane integrity of the cell [45]. Results of total (viable plus non-viable) and viable cell counts were expressed as log cell•cm −2 .…”

Incorporation of carbon nanotubes in polydimethylsiloxane to control Escherichia coli adhesion

“…It was also shown that its biofilm formation behavior is similar to other E. coli strains including a clinical isolate that is often used for antimicrobial susceptibility tests [43]. Additionally, this strain was successfully used to produce a model recombinant protein in a biofilm flow cell system [16][17][18].…”

Comparing the Recombinant Protein Production Potential of Planktonic and Biofilm Cells

“…A reactor system comprising a recirculating tank (for planktonic cells), a vertical flow cell reactor (where biofilms are formed), and peristaltic and centrifuge pumps that allow the circulation of the bacterial suspension was operated as described by Gomes et al [ 25 ]. The flow cell reactor is a semi-circular Perspex duct with apertures on its flat wall to fit removable Perspex pieces (coupons) to which polyvinyl chloride (PVC) slides were glued.…”

“…E. coli cells were grown by recirculating the bacterial suspension at 30 °C for 11 days under turbulent flow (Reynolds number of 4600) [ 25 ]. The recirculating tank was aerated using an air pump (flow rate of 108 L h −1 ) and continuously fed with lysogeny medium (LB-Miller; Sigma, St. Louis, USA) supplemented with 20 μg mL –1 kanamycin at a flow rate of 0.025 L h –1 [ 25 ].…”

“…Biofilm total (viable plus non-viable) and viable cell counts were determined using the Live/Dead ® BacLight TM bacterial viability kit (Syto9/propidium iodide; Invitrogen Life Technologies, Alfagene, Carcavelos, Portugal) as fully described in Gomes et al [ 25 ]. Briefly, bacterial cells from the biofilm suspension were filtered through a Nucleopore Track-Etch Membrane of black polycarbonate (pore size of 0.22 µm; Whatman Ltd., Buckinghamshire, UK), stained for 10 min in the dark and observed using a Leica DM LB2 epifluorescence microscope connected to a Leica DFC300 FX camera (Leica Microsystems Ltd, Heerbrugg, Switzerland).…”