Synthetic Biology Experimental Design Lab

Learning Outcomes

Upon successful completion of the course, students are expected to:

• understand the theoretical principles underlying core laboratory techniques used in synthetic biology
• explain the main stages of recombinant plasmid construction and molecular cloning
• describe the principles of mammalian cell culture and transient gene expression
• understand the basis of fluorescence labeling, microscopy, and image-based cellular analysis
• explain the principles and applications of flow cytometry for single-cell analysis
• recognize the role of controls, quality assessment, and data interpretation in experimental workflows
• understand how molecular, cellular, imaging, and cytometric methods are integrated in synthetic biology research

In addition, students will have acquired the following skills:

• apply fundamental laboratory techniques in molecular biology
• perform basic mammalian cell culture procedures
• prepare and analyze biological samples using fluorescence-based imaging techniques
• perform cell sample preparation and data acquisition using flow cytometry.
• apply the Design-Build-Test-Learn (DBTL) workflow to practical laboratory experiments, from plasmid construction to gene expression analysis in mammalian cells.

Finally, students will demonstrate the following competencies:

• analyse, evaluate and interpret experimental data obtained from molecular, cellular, and imaging experiments
• communicate experimental procedures and results effectively

Module Syllabus

This course provides comprehensive practical training in the fundamental laboratory techniques required in synthetic biology. Emphasis is given on the development of hands-on experimental skills and the interpretation of experimental data. Through a series of guided laboratory sessions, students gain practical experience in molecular manipulation, cell culture techniques, imaging technologies, and flow cytometry methods.

The laboratory exercises program includes the following modules:

Module 1 –Cloning and Plasmid Construction

The course begins with training in molecular cloning techniques. Students design and perform Polymerase Chain Reaction (PCR) to amplify the full-length cDNA of a human nuclear protein, restriction enzyme digestion, DNA fragment purification, and cloning into eukaryotic expression plasmid vectors designed to produce proteins fused to green fluorescent protein (GFP), ligation reactions, bacterial transformation, and the use of selection and screening strategies to identify recombinant clones.

• Exercise 1: Introduction to Molecular Cloning and Experimental Design (Safety and laboratory procedures, overview of the cloning strategy, primer design, plasmid vectors and cloning sites)
• Exercise 2: PCR Amplification of Target cDNA
• Exercise 3: Gel electrophoresis and Restriction Digestion
• Exercise 4: DNA Purification and Ligation
• Exercise 5: Bacterial Transformation
• Exercise 6: Screening Recombinant Clones

Module 2 – Mammalian Cell Culture and Transfection

The second part of the course focuses on cell culture techniques. Students are introduced to standard practices for maintaining and manipulating cultured cells, biosafety principles, sterile techniques, and laboratory waste management. This module also includes transient transfection methods.

• Exercise 7: Introduction to Cell Culture (Biosafety procedures, sterile technique, media preparation and cell handling)
• Exercise 8 – Cell Maintenance and Quality Control (Subculturing and passaging cells, monitoring cell morphology, cell counting)
• Exercise 9 – Transient Transfection (Transfection of HeLa cells with the recombinant GFP plasmid, optimization of transfection conditions)

Module 3 – Fluorescence and Confocal Microscopy

The course further provides practical training in light and fluorescence microscopy. Students learn specimen preparation techniques, the use of fluorescent probes for labeling cellular components, the operation of fluorescence and confocal microscopes, as well as the use of image-analysis software for quantitative analysis.

• Exercise 10: Immunofluorescence Staining (Specimen preparation: Fixation and permeabilization of transfected cells; incubation of the cells with specific fluorescence probes to visualize subcellular organelles e.g. DAPI for nuclear staining; indirect immunofluorescence analysis using specific antibodies against nuclear lamins and fluorescence labeled secondary antibodies).
• Exercise 11 – Confocal Laser Scanning Microscopy, CLSM: Introduction to CLSM; Single section, multi-channel Image acquisition, Quantitative Image Analysis. 

Module 4 – Flow Cytometry

Finally, the course includes training in flow cytometry. Students gain experience in sample preparation, instrument setup, and data acquisition. They learn how to acquire and interpret cytometric data and apply gating strategies to identify and quantify specific cell populations.

• Exercise 12 – Cytometry Sample Preparation, Data Acquisition and Analysis, determination of transfection efficiency, apply gating strategies to identify GFP-positive cells.

Module 5 – Presentation of the results

At the end of the laboratory course, students present the results obtained from their experimental work. Students also analyze and interpret their results, discuss possible inconsistencies in the experimental results, and their outcomes compared to the expected observations.

Suggested Bibliography

• Brown, T. A. (2020). Gene Cloning and DNA Analysis: An Introduction (8th ed.). Wiley-Blackwell.
• Green, M. R., & Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor Laboratory Press.
• Freshney, R. I. (2016). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (7th ed.). Wiley-Blackwell.
• Murphy, D. B., & Davidson, M. W. (2012). Fundamentals of Light Microscopy and Electronic Imaging (2nd ed.). Wiley-Blackwell.
• Pawley, J. B. (Ed.). (2006). Handbook of Biological Confocal Microscopy (3rd ed.). Springer.
• Givan, A. L. (2013). Flow Cytometry: First Principles (2nd ed.). Wiley-Blackwell.