Understanding the flow behavior around marine biofilms.

In vitro platforms capable of mimicking the hydrodynamic conditions prevailing in natural aquatic environments have been previously validated and used to predict the fouling behavior on different surfaces. Computational Fluid Dynamics (CFD) has been used to predict the shear forces occurring in these platforms. In general, these predictions are made for the initial stages of biofilm formation, where the amount of biofilm does not affect the flow behavior, enabling the estimation of the shear forces that initial adhering organisms have to withstand. In this work, we go a step further in understanding the flow behavior when a mature biofilm is present in such platforms to better understand the shear rate distribution affecting marine biofilms. Using 3D images obtained by Optical Coherence Tomography, a mesh was produced and used in CFD simulations. Biofilms of two different marine cyanobacteria were developed in agitated microtiter plates incubated at two different shaking frequencies for 7 weeks. The biofilm-flow interactions were characterized in terms of the velocity field and shear rate distribution. Results show that global hydrodynamics imposed by the different shaking frequencies affect biofilm architecture and also that this architecture affects local hydrodynamics, causing a large heterogeneity in the shear rate field. Biofilm cells located in the streamers of the biofilm are subjected to much higher shear values than those located on the bottom of the streamers and this dispersion in shear rate values increases at lower bulk fluid velocities. This heterogeneity in the shear force field may be a contributing factor for the heterogeneous behavior in metabolic activity, growth status, gene expression pattern, and antibiotic resistance often associated with nutrient availability within the biofilm.

The use of biomimetic surfaces to reduce single- and dual-species biofilms of Escherichia coli and Pseudomonas putida.

The ability of bacteria to adhere to and form biofilms on food contact surfaces poses serious challenges, as these may lead to the cross-contamination of food products. Biomimetic topographic surface modifications have been explored to enhance the antifouling performance of materials. In this study, the topography of two plant leaves, Brassica oleracea var. botrytis (cauliflower, CF) and Brassica oleracea capitate (white cabbage, WC), was replicated through wax moulding, and their antibiofilm potential was tested against single- and dual-species biofilms of Escherichia coli and Pseudomonas putida. Biomimetic surfaces exhibited higher roughness values (SaWC = 4.0 ± 1.0 µm and SaCF = 3.3 ± 1.0 µm) than the flat control (SaF = 0.6 ± 0.2 µm), whilst the CF surface demonstrated a lower interfacial free energy (ΔGiwi) than the WC surface (-100.08 mJ m-2 and -71.98 mJ m-2, respectively). The CF and WC surfaces had similar antibiofilm effects against single-species biofilms, achieving cell reductions of approximately 50% and 60% for E. coli and P. putida, respectively, compared to the control. Additionally, the biomimetic surfaces led to reductions of up to 60% in biovolume, 45% in thickness, and 60% in the surface coverage of single-species biofilms. For dual-species biofilms, only the E. coli strain growing on the WC surface exhibited a significant decrease in the cell count. However, confocal microscopy analysis revealed a 60% reduction in the total biovolume and surface coverage of mixed biofilms developed on both biomimetic surfaces. Furthermore, dual-species biofilms were mainly composed of P. putida, which reduced E. coli growth. Altogether, these results demonstrate that the surface properties of CF and WC biomimetic surfaces have the potential for reducing biofilm formation.

Antibiofilm Effect of Nitric Acid-Functionalized Carbon Nanotube-Based Surfaces against E. coli and S. aureus.

Chemically modified carbon nanotubes are recognized as effective materials for tackling bacterial infections. In this study, pristine multi-walled carbon nanotubes (p-MWCNTs) were functionalized with nitric acid (f-MWCNTs), followed by thermal treatment at 600 °C, and incorporated into a poly(dimethylsiloxane) (PDMS) matrix. The materials' textural properties were evaluated, and the roughness and morphology of MWCNT/PDMS composites were assessed using optical profilometry and scanning electron microscopy, respectively. The antibiofilm activity of MWCNT/PDMS surfaces was determined by quantifying culturable Escherichia coli and Staphylococcus aureus after 24 h of biofilm formation. Additionally, the antibacterial mechanisms of MWCNT materials were identified by flow cytometry, and the cytotoxicity of MWCNT/PDMS composites was tested against human kidney (HK-2) cells. The results revealed that the antimicrobial activity of MWCNTs incorporated into a PDMS matrix can be efficiently tailored through nitric acid functionalization, and it can be increased by up to 49% in the absence of surface carboxylic groups in f-MWCNT samples heated at 600 °C and the presence of redox activity of carbonyl groups. MWCNT materials changed the membrane permeability of both Gram-negative and Gram-positive bacteria, while they only induced the production of ROS in Gram-positive bacteria. Furthermore, the synthesized composites did not impact HK-2 cell viability, confirming the biocompatibility of MWCNT composites.

Graphene-Based Composites for Biomedical Applications: Surface Modification for Enhanced Antimicrobial Activity and Biocompatibility.

The application of graphene-based materials in medicine has led to significant technological breakthroughs. The remarkable properties of these carbon materials and their potential for functionalization with various molecules and compounds make them highly attractive for numerous medical applications. To enhance their functionality and applicability, extensive research has been conducted on surface modification of graphene (GN) and its derivatives, including modifications with antimicrobials, metals, polymers, and natural compounds. This review aims to discuss recent and relevant studies related to advancements in the formulation of graphene composites, addressing their antimicrobial and/or antibiofilm properties and evaluating their biocompatibility, with a primary focus on their biomedical applications. It was concluded that GN surface modification, particularly with compounds intrinsically active against bacteria (e.g., antimicrobial peptides, silver and copper nanomaterials, and chitosan), has resulted in biomaterials with improved antimicrobial performance. Furthermore, the association of GN materials with non-natural polymers provides composites with increased biocompatibility when interfaced with human tissues, although with slightly lower antimicrobial efficacy. However, it is crucial to highlight that while modified GN materials hold huge potential, their widespread use in the medical field is still undergoing research and development. Comprehensive studies on safety, long-term effects, and stability are essential before their adoption in real-world medical scenarios.

Exploring Nitrogen-Functionalized Graphene Composites for Urinary Catheter Applications.

Graphene has been broadly studied, particularly for the fabrication of biomedical devices, owing to its physicochemical and antimicrobial properties. In this study, the antibiofilm efficacy of graphene nanoplatelet (GNP)-based composites as coatings for urinary catheters (UCs) was investigated. GNPs were functionalized with nitrogen (N-GNP) and incorporated into a polydimethylsiloxane (PDMS) matrix. The resulting materials were characterized, and the N-GNP/PDMS composite was evaluated against single- and multi-species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Both biofilm cell composition and structure were analyzed. Furthermore, the antibacterial mechanisms of action of N-GNP were explored. The N-GNP/PDMS composite showed increased hydrophobicity and roughness compared to PDMS. In single-species biofilms, this composite significantly reduced the number of S. aureus, P. aeruginosa, and K. pneumoniae cells (by 64, 41, and 29%, respectively), and decreased S. aureus biofilm culturability (by 50%). In tri-species biofilms, a 41% reduction in total cells was observed. These results are aligned with the outcomes of the biofilm structure analysis. Moreover, N-GNP caused changes in membrane permeability and triggered reactive oxygen species (ROS) synthesis in S. aureus, whereas in Gram-negative bacteria, it only induced changes in cell metabolism. Overall, the N-GNP/PDMS composite inhibited biofilm development, showing the potential of these carbon materials as coatings for UCs.

Effect of Hydrogen Peroxide on Cyanobacterial Biofilms.

Although a range of disinfecting formulations is commercially available, hydrogen peroxide is one of the safest chemical agents used for disinfection in aquatic environments. However, its effect on cyanobacterial biofilms is poorly investigated. In this work, biofilm formation by two filamentous cyanobacterial strains was evaluated over seven weeks on two surfaces commonly used in marine environments: glass and silicone-based paint (Sil-Ref) under controlled hydrodynamic conditions. After seven weeks, the biofilms were treated with a solution of hydrogen peroxide (H2O2) to assess if disinfection could affect long-term biofilm development. The cyanobacterial biofilms appeared to be tolerant to H2O2 treatment, and two weeks after treatment, the biofilms that developed on glass by one of the strains presented higher biomass amounts than the untreated biofilms. This result emphasizes the need to correctly evaluate the efficiency of disinfection in cyanobacterial biofilms, including assessing the possible consequences of inefficient disinfection on the regrowth of these biofilms.

Insights into the antimicrobial properties of a cationic steroid and antibiofilm performance in PDMS-based coatings to potentially treat urinary infections.

Currently, multidrug-resistant (MDR) infections are one of the most important threats, driving the search for new antimicrobial compounds. Cationic peptide antibiotics (CPAs) and ceragenins (CSAs) contain in their structures cationic groups and adopt a facially amphiphilic conformation, conferring the ability to permeate the membranes of bacteria and fungi. Keeping these features in mind, an amine steroid, DOCA-NH2, was found to be active against reference strains and MDR isolates of Gram-positive Enterococcus faecalis and Staphylococcus aureus and Gram-negative Escherichia coli and Pseudomonas aeruginosa. The compound was active against all the tested microorganisms, having bactericidal and fungicidal activity, displaying minimal inhibitory concentrations (MICs) between 16 and 128 µg mL-1. No synergy with clinically relevant antibacterial drugs was found. However, the compound was able to completely inhibit the biofilm formation of bacteria exposed to the MIC of the compound. For E. coli and E. faecalis, inhibition of biofilm formation occurred at half the MIC. Besides, DOCA-NH2 inhibited the dimorphic transition of Candida albicans at concentrations 4 times lower than the MIC, and can reduce the microorganism virulence and biofilm formation was significantly reduced at both MIC and half the MIC. Polydimethylsiloxane-based coatings containing DOCA-NH2 (0.5, 1.0, and 1.5 wt%) were prepared and tested against the E. coli biofilm formation under hydrodynamic conditions similar to those prevailing in ureteral stents. A biofilm reduction of approximately 80% was achieved when compared to the control.

Development of Antifouling Strategies for Marine Applications.

Marine biofouling is an undeniable challenge for aquatic systems since it is responsible for several environmental and ecological problems and economic losses. Several strategies have been developed to mitigate fouling-related issues in marine environments, including developing marine coatings using nanotechnology and biomimetic models, and incorporating natural compounds, peptides, bacteriophages, or specific enzymes on surfaces. The advantages and limitations of these strategies are discussed in this review, and the development of novel surfaces and coatings is highlighted. The performance of these novel antibiofilm coatings is currently tested by in vitro experiments, which should try to mimic real conditions in the best way, and/or by in situ tests through the immersion of surfaces in marine environments. Both forms present their advantages and limitations, and these factors should be considered when the performance of a novel marine coating requires evaluation and validation. Despite all the advances and improvements against marine biofouling, progress toward an ideal operational strategy has been slow given the increasingly demanding regulatory requirements. Recent developments in self-polishing copolymers and fouling-release coatings have yielded promising results which set the basis for the development of more efficient and eco-friendly antifouling strategies.

Use of Probiotics to Control Biofilm Formation in Food Industries.

Microorganisms tend to adhere to food contact surfaces and form biofilms, which serve as reservoirs for bacteria that can contaminate food. As part of a biofilm, bacteria are protected from the stressful conditions found during food processing and become tolerant to antimicrobials, including traditional chemical sanitisers and disinfectants. Several studies in the food industry have shown that probiotics can prevent attachment and the consequent biofilm formation by spoilage and pathogenic microorganisms. This review discusses the most recent and relevant studies on the effects of probiotics and their metabolites on pre-established biofilms in the food industry. It shows that the use of probiotics is a promising approach to disrupt biofilms formed by a large spectrum of foodborne microorganisms, with Lactiplantibacillus and Lacticaseibacillus being the most tested genera, both in the form of probiotic cells and as sources of cell-free supernatant. The standardisation of anti-biofilm assays for evaluating the potential of probiotics in biofilm control is of extreme importance, enabling more reliable, comparable, and predictable results, thus promoting significant advances in this field.

Graphene-Based Coating to Mitigate Biofilm Development in Marine Environments.

Due to its several economic and ecological consequences, biofouling is a widely recognized concern in the marine sector. The search for non-biocide-release antifouling coatings has been on the rise, with carbon-nanocoated surfaces showing promising activity. This work aimed to study the impact of pristine graphene nanoplatelets (GNP) on biofilm development through the representative marine bacteria Cobetia marina and to investigate the antibacterial mechanisms of action of this material. For this purpose, a flow cytometric analysis was performed and a GNP/polydimethylsiloxane (PDMS) surface containing 5 wt% GNP (G5/PDMS) was produced, characterized, and assessed regarding its biofilm mitigation potential over 42 days in controlled hydrodynamic conditions that mimic marine environments. Flow cytometry revealed membrane damage, greater metabolic activity, and endogenous reactive oxygen species (ROS) production by C. marina when exposed to GNP 5% (w/v) for 24 h. In addition, C. marina biofilms formed on G5/PDMS showed consistently lower cell count and thickness (up to 43% reductions) than PDMS. Biofilm architecture analysis indicated that mature biofilms developed on the graphene-based surface had fewer empty spaces (34% reduction) and reduced biovolume (25% reduction) compared to PDMS. Overall, the GNP-based surface inhibited C. marina biofilm development, showing promising potential as a marine antifouling coating.

Development of Cork Biocomposites Enriched with Chitosan Targeting Antibacterial and Antifouling Properties.

The demand for bio-based and safer composite materials is increasing due to the growth of the industry, human population, and environmental concerns. In this framework, sustainable and safer cork-polymer composites (CPC), based on green low-density polyethylene (LDPE) were developed using melt-based technologies. Chitosan and polyethylene-graft-maleic anhydride (PE-g-MA) were employed to enhance the CPC's properties. The morphology, wettability, mechanical, thermal, and antibacterial properties of the CPC against Pseudomonas putida (P. putida) and Staphylococcus aureus (S. aureus) were examined. The CPC showed improved stiffness when compared with that of the LDPE matrix, preferably when combined with chitosan and PE-g-MA (5 wt. %), reinforcing the stiffness (58.8%) and the strength (66.7%). Chitosan also increased the composite stiffness and strength, as well as reduced the surface hydrophilicity. The CPCs' antibacterial activity revealed that cork significantly reduces the biofilm on the polymer matrix. The highest biofilm reduction was found with CPC containing cork and 5 wt. % chitosan for both P. putida (54% reduction) and S. aureus (36% reduction), confirming their potential to extend the lifespan of products for packaging and healthcare, among other applications. This work leads to the understanding of the factors that influence biofilm formation in cork composites and provides a strategy to reinforce their behavior using chitosan.

Characterization and biofouling potential analysis of two cyanobacterial strains isolated from Cape Verde and Morocco.

Cyanobacteria are new sources of value-added compounds but also ubiquitous and harmful microfoulers on marine biofouling. In this work, the isolation and identification of two cyanobacterial strains isolated from Cape Verde and Morocco, as well as their biofilm-forming ability on glass and Perspex under controlled hydrodynamic conditions, were performed. Phylogenetic analysis revealed that cyanobacterial strains isolated belong to Leptothoe and Jaaginema genera (Leptothoe sp. LEGE 181153 and Jaaginema sp. LEGE 191154). From quantitative and qualitative data of wet weight, chlorophyll a content and biofilm thickness obtained by optical coherence tomography, no significant differences were found in biofilms developed by the same cyanobacterial strain on different surfaces (glass and Perspex). However, the biofilm-forming potential of Leptothoe sp. LEGE 181153 proved to be higher compared with Jaaginema sp. LEGE 191154, particularly at the maturation stage of biofilm development. Three-dimensional biofilm images obtained from confocal laser scanning microscopy showed different patterns between both cyanobacterial strains and also among the two surfaces. Because standard methodologies to evaluate cyanobacterial biofilm formation, as well as two different optical imaging techniques, were used, this work also highlights the possibility of integrating different techniques to evaluate a complex phenomenon like cyanobacterial biofilm development.

Assessment of the Antibiofilm Performance of Chitosan-Based Surfaces in Marine Environments.

Marine biofouling is a natural process often associated with biofilm formation on submerged surfaces, creating a massive economic and ecological burden. Although several antifouling paints have been used to prevent biofouling, growing ecological concerns emphasize the need to develop new and environmentally friendly antifouling approaches such as bio-based coatings. Chitosan (CS) is a natural polymer that has been widely used due to its outstanding biological properties, including non-toxicity and antimicrobial activity. This work aims to produce and characterize poly (lactic acid) (PLA)-CS surfaces with CS of different molecular weight (Mw) at different concentrations for application in marine paints. Loligo opalescens pens, a waste from the fishery industry, were used as a CS source. The antimicrobial activity of the CS and CS-functionalized surfaces was assessed against Cobetia marina, a model proteobacterium for marine biofouling. Results demonstrate that CS targets the bacterial cell membrane, and PLA-CS surfaces were able to reduce the number of culturable cells up to 68% compared to control, with this activity dependent on CS Mw. The antifouling performance was corroborated by Optical Coherence Tomography since PLA-CS surfaces reduced the biofilm thickness by up to 36%, as well as the percentage and size of biofilm empty spaces. Overall, CS coatings showed to be a promising approach to reducing biofouling in marine environments mimicked in this work, contributing to the valorization of fishing waste and encouraging further research on this topic.

Hydrodynamic conditions affect the proteomic profile of marine biofilms formed by filamentous cyanobacterium.

Proteomic studies on cyanobacterial biofilms can be an effective approach to unravel metabolic pathways involved in biofilm formation and, consequently, obtain more efficient biofouling control strategies. Biofilm development by the filamentous cyanobacterium Toxifilum sp. LEGE 06021 was evaluated on different surfaces, glass and perspex, and at two significant shear rates for marine environments (4 s-1 and 40 s-1). Higher biofilm development was observed at 4 s-1. Overall, about 1877 proteins were identified, and differences in proteome were more noticeable between hydrodynamic conditions than those found between surfaces. Twenty Differentially Expressed Proteins (DEPs) were found between 4 s-1 vs. 40 s-1. On glass, some of these DEPs include phage tail proteins, a carotenoid protein, cyanophynase glutathione-dependent formaldehyde dehydrogenase, and the MoaD/ThiS family protein, while on perspex, DEPs include transketolase, dihydroxy-acid dehydratase, iron ABC transporter substrate-binding protein and protein NusG. This study contributes to developing a standardized protocol for proteomic analysis of filamentous cyanobacterial biofilms. This kind of proteomic analysis can also be useful for different research fields, given the broad spectrum of promising secondary metabolites and added-value compounds produced by cyanobacteria, as well as for the development of new antibiofilm strategies.

The Use of 3D Optical Coherence Tomography to Analyze the Architecture of Cyanobacterial Biofilms Formed on a Carbon Nanotube Composite.

The development of environmentally friendly antifouling strategies for marine applications is of paramount importance, and the fabrication of innovative nanocomposite coatings is a promising approach. Moreover, since Optical Coherence Tomography (OCT) is a powerful imaging technique in biofilm science, the improvement of its analytical power is required to better evaluate the biofilm structure under different scenarios. In this study, the effect of carbon nanotube (CNT)-modified surfaces in cyanobacterial biofilm development was assessed over a long-term assay under controlled hydrodynamic conditions. Their impact on the cyanobacterial biofilm architecture was evaluated by novel parameters obtained from three-dimensional (3D) OCT analysis, such as the contour coefficient, total biofilm volume, biovolume, volume of non-connected pores, and the average size of non-connected pores. The results showed that CNTs incorporated into a commercially used epoxy resin (CNT composite) had a higher antifouling effect at the biofilm maturation stage compared to pristine epoxy resin. Along with a delay in biofilm development, a decrease in biofilm wet weight, thickness, and biovolume was also achieved with the CNT composite compared to epoxy resin and glass (control surfaces). Additionally, biofilms developed on the CNT composite were smoother and presented a lower porosity and a strictly packed structure when compared with those formed on the control surfaces. The novel biofilm parameters obtained from 3D OCT imaging are extremely important when evaluating the biofilm architecture and behavior under different scenarios beyond marine applications.

Advances on Bacterial and Fungal Biofilms for the Production of Added-Value Compounds.

In recent years, abundant research has been performed on biofilms for the production of compounds with biotechnological and industrial relevance. The use of biofilm platforms has been seen as a compelling approach to producing fine and bulk chemicals such as organic acids, alcohols, and solvents. However, the production of recombinant proteins using this system is still scarce. Biofilm reactors are known to have higher biomass density, operational stability, and potential for long-term operation than suspended cell reactors. In addition, there is an increasing demand to harness industrial and agricultural wastes and biorefinery residues to improve process sustainability and reduce production costs. The synthesis of recombinant proteins and other high-value compounds is mainly achieved using suspended cultures of bacteria, yeasts, and fungi. This review discusses the use of biofilm reactors for the production of recombinant proteins and other added-value compounds using bacteria and fungi.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

Antifouling Performance of Carbon-Based Coatings for Marine Applications: A Systematic Review.

Although carbon materials are widely used in surface engineering, particularly graphene (GP) and carbon nanotubes (CNTs), the application of these nanocomposites for the development of antibiofilm marine surfaces is still poorly documented. The aim of this study was, thus, to gather and discuss the relevant literature concerning the antifouling performance of carbon-based coatings against marine micro- and macrofoulers. For this purpose, a PRISMA-oriented systematic review was conducted based on predefined criteria, which resulted in the selection of thirty studies for a qualitative synthesis. In addition, the retrieved publications were subjected to a quality assessment process based on an adapted Methodological Index for Non-Randomized Studies (MINORS) scale. In general, this review demonstrated the promising antifouling performance of these carbon nanomaterials in marine environments. Further, results from the revised studies suggested that functionalized GP- and CNTs-based marine coatings exhibited improved antifouling performance compared to these materials in pristine forms. Thanks to their high self-cleaning and enhanced antimicrobial properties, as well as durability, these functionalized composites showed outstanding results in protecting submerged surfaces from the settlement of fouling organisms in marine settings. Overall, these findings can pave the way for the development of new carbon-engineered surfaces capable of preventing marine biofouling.

Hydrodynamic Effects on Biofilm Development and Recombinant Protein Expression.

Hydrodynamics play an important role in the rate of cell attachment and nutrient and oxygen transfer, which can affect biofilm development and the level of recombinant protein production. In the present study, the effects of different flow conditions on the development of Escherichia coli biofilms and the expression of a model recombinant protein (enhanced green fluorescent protein, eGFP) were examined. Planktonic and biofilm cells were grown at two different flow rates in a recirculating flow cell system for 7 days: 255 and 128 L h-1 (corresponding to a Reynolds number of 4600 and 2300, respectively). The fluorometric analysis showed that the specific eGFP production was higher in biofilms than in planktonic cells under both hydrodynamic conditions (3-fold higher for 255 L h-1 and 2-fold higher for 128 L h-1). In the biofilm cells, the percentage of eGFP-expressing cells was on average 52% higher at a flow rate of 255 L h-1. Furthermore, a higher plasmid copy number (PCN) was obtained for the highest flow rate for both planktonic (244 PCN/cell versus 118 PCN/cell) and biofilm cells (43 PCN/cell versus 29 PCN/cell). The results suggested that higher flow velocities promoted eGFP expression in E. coli biofilms.

Implementation of a Practical Teaching Course on Protein Engineering.

Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli-the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.