Molecular Biology and Biotechnology Laboratory A 1200-1CHMLBMAL5

Molecular Biology and Biotechnology Laboratory is an integral component of the “Medical Biotechnology” course, designed to provide students with hands-on experience in contemporary molecular biology techniques and their applications in both industrial and medical biotechnology. Laboratory classes are organized as one- or two-day modules, allowing students to gradually develop competencies and gain a deeper understanding of the discussed topics.

During the course, students will become familiar with aseptic techniques, microbial cultivation and transformation, plasmid and protein isolation, biological activity assays, as well as the basics of diagnostic system and biomaterial design. The course emphasizes the practical application of acquired knowledge — in diagnostics and in the development of advanced therapeutic systems such as liposomal drug carriers or microfluidic platforms. The laboratory combines knowledge from biology, chemistry, physical chemistry, and materials engineering, providing students with comprehensive training for careers in life sciences, biomedicine, and modern pharmaceutical technologies.

Bacterial cell cultivation on liquid and solid media:
Students learn the principles of aseptic technique and prepare their own bacterial media. They perform streak plating and serial dilutions to determine colony-forming units (CFU) and the correlation between optical density and bacterial count. Bacterial growth is analyzed on different media types, and growth curves are constructed. The exercise also includes microbiological purity assessment of the surrounding environment based on environmental sampling. This serves as a practical introduction to quality control in microbiological biotechnology.

Determination of Minimum Inhibitory Concentration (MIC):
Students assess the antimicrobial activity of selected substances using the disc diffusion method (Kirby-Bauer), widely used in clinical diagnostics and antibiotic evaluation. They perform serial dilutions of compounds (antibiotics, metal salts, plant extracts, nanoparticles) and analyze their effect on E. coli growth. Based on the diameter of inhibition zones, they estimate MIC values, learning to interpret results per EUCAST guidelines. The exercise demonstrates the use of semi-quantitative assays in pharmaceutical screening and microbial resistance analysis, emphasizing critical evaluation of biological activity.

Plasmid isolation from E. coli under selective conditions:
Students compare plasmid DNA yields from E. coli cultured with and without antibiotics, illustrating the importance of selection in maintaining genetic constructs. Plasmids are isolated via classical or column-based methods, then analyzed using electrophoresis and spectrophotometry. Enzymatic digestion is performed to assess insert integrity and plasmid copy number. This introduces students to the fundamentals of genetic engineering and the preparation of materials for protein expression, vaccine production, biosensors, or biologic drugs.

Modification of liposomes using recombinant protein:
Students transform E. coli with a pGLO plasmid to produce GFP protein, which is purified via affinity chromatography. The protein is used to functionalize liposome surfaces, creating a fluorescent nanocarrier model. The exercise introduces protein engineering and the design of nanosystems for drug delivery, demonstrating applications in targeted therapies, vaccines, and nanosensors. It bridges molecular biology techniques with therapeutic structure design in nanobiotechnology and pharmaceutical sciences.

Enzyme-linked Immunosorbent Assay (ELISA):
Students perform an indirect ELISA to detect a specific antigen or antibody, learning each step: antigen binding, blocking, secondary antibody binding, and colorimetric detection. Test validation and the role of controls are discussed. Applications in infectious disease diagnostics, allergy testing, immune response analysis, and vaccine evaluation are covered. The practical session also highlights assay limitations and mitigation strategies.

Lateral Flow Assay (LFA):
Students perform a rapid diagnostic test based on lateral flow, using in-house synthesized gold nanoparticles conjugated with antibodies. They prepare a basic detection strip (nitrocellulose membrane with test and control lines) to detect a chosen analyte. The exercise demonstrates the principles, design, and limitations of rapid tests (e.g., pregnancy or COVID-19 tests), including nanoparticle stability and signal interpretation. Applications in point-of-care diagnostics are emphasized.

Protein immunoprecipitation using antibodies:
Students isolate specific proteins from biological samples using antibody-coated magnetic beads. They design buffers, prepare samples, and elute immune complexes. The resulting samples are analyzed via electrophoresis. This technique is applied in protein–protein interaction studies, gene expression analysis, and biologic drug validation. The exercise emphasizes purification of recombinant or endogenous target proteins.

Bacterial bioprinting in hydrogel matrix:
Students create living biological material from bacterial cultures and hydrogels for 3D bioprinting. They prepare hydrogel matrices with embedded cells and design simple geometric models for printing. Printed structures are assessed for cell viability and material stability. The exercise introduces live biomaterial creation and tissue engineering systems, with applications in biosensors, wound dressings, and therapeutic devices.

Biotechnological production processes using microorganisms:
Students work with two types of yeast: Saccharomyces cerevisiae and Torula, known for their industrial applications. They cultivate cells on various media, then perform biomass separation and analyze macromolecular fractions (polysaccharides, proteins). Purification techniques like dialysis and ultrafiltration are applied, and industrial use of enzymes and metabolites is discussed. This exercise links lab work with real-world biotechnological applications, including scale-up challenges and differences between molecular and technological approaches.

Secondary processing of microbial biomass in biotechnology:
This session focuses on the second stage of microbial biomass processing. Students obtain yeast extract enriched with nucleotides and a β-glucan-containing fraction. They use physicochemical cell disruption methods, followed by fractionation and purification. The importance of mixture separation, process standardization, and parameter control is emphasized. Special attention is given to analytical evaluation of extracts using spectrophotometric and comparative methods. The exercise shows how microorganisms can yield valuable components for supplements, immunomodulators, and drug carriers, illustrating quality optimization and product validation strategies in biotech.