Teaching plan for the course unit

 

 
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General information

 

Course unit name: Laboratory III

Course unit code: 361577

Academic year: 2020-2021

Coordinator: Joan Carles Ferrer Artigas

Department: Department of Biochemistry and Molecular Biomedicine

Credits: 6

Single program: S

 

 

Estimated learning time

Total number of hours 150

 

Face-to-face and/or online activities

71
 

-  Lecture with practical component

Face-to-face

  

17
 

-  Laboratory session

Face-to-face

  

54

Supervised project

39

Independent learning

40

 

 

Competences to be gained during study

 

   -

Capacity for learning and responsibility (capacity for analysis and synthesis, to adopt global perspectives and to apply the knowledge acquired/capacity to take decisions and adapt to new situations).
   -

Ability to work in a team (capacity to collaborate with others and contribute to a common project/capacity to work in cross-disciplinary and multicultural teams).
   -

Capacity to work safely in a laboratory environment.
   -

Understanding of life processes based on the cellular and molecular study of organisms.
   -

Capacity to understand and explain the underlying chemical principles of biochemical reactions and techniques.
   -

Capacity to design, plant, carry out and evaluate experiments and research projects.
   -

Skills to apply instrumental, analytical and molecular techniques.
   -

Capacity to separate substances isolated from living cells and determining their structures, chemical properties and functional properties.
   -

Capacity to combine substances isolated from living cells under controlled conditions, to determine how they react and what results are obtained.
   -

Ability to prepare cell, tissue and microorganism cultures.

Learning objectives

 

Referring to knowledge

— Understand the basic concepts behind the tasks undertaken in a biochemistry laboratory.

— Acquire theoretical and practical knowledge of the techniques commonly used in a biochemistry laboratory. More specifically: learn to express and isolate enzymes and other proteins from bacterial cultures; purify nucleic acids form tissues; amplify specific DNA fragments through PCR; modify DNA sequences by site-directed mutagenesis; perform physicochemical analyses of proteins and DNA; characterise enzymes kinetically; perform computer-assisted analyses of protein sequences and structures and DNA primers.

 

Referring to abilities, skills

— Learn to relate and integrate concepts.

— Design, perform and analyse basic experimental protocols.

— Learn how to keep a laboratory notebook.

— Be able to comment experimental results and present them in public.

— Understand and follow simple projects of biochemical, biomedical and biotechnological research.

— Intervene in basic aspects of these projects.

— Use statistical and computer tools to analyse experimental data.

 

Referring to attitudes, values and norms

— Learn to work in groups in the laboratory.

— Acquire study and planning habits.

— Know and follow the general safety rules of a biochemistry laboratory and those specific to the products and instruments used.

 

 

Teaching blocks

 

1. Introduction

*   

1.1. Presentation of the course programme; Teaching methodology and assessment procedure

1.2. Organisation of the course; Distribution of practical sessions and groups

2. Recombinant DNA techniques

*   

2.1. Isolation and purification of DNA and RNA

2.2. DNA libraries; Types and construction of DNA libraries

2.3. Foundations of the polymerase chain reaction (PCR)

2.4. Oligonucleotide site directed mutagenesis

2.5. DNA sequencing

3. Heterologous expression of proteins

*   

3.1. Systems of expression of recombinant proteins

3.2. Expression in prokaryotes

3.3. Vectors of prokaryote expression; Fusion proteins; Handles of purification

3.4. Extraction and purification of recombinant proteins

3.5. Troubleshooting and optimisation of results

4. Chromatographic techniques II

*   

4.1. Overview of gel filtration and ionic exchange chromatography

4.2. Affinity chromatography

4.3. Metal-chelate affinity chromatography

4.4. Hydrophobic interaction chromatography (HIC)

5. Electrophoretic techniques II

*   

5.1. Electrophoresis of nucleic acids in agarose gels

5.2. Electrophoresis in DNA sequencing

5.3. Polyacrylamide gel electrophoresis under native (PAGE) and denaturing conditions (SDS-PAGE); General and specific protein staining

5.4. Isoelectrofocusing

5.5. Two-dimensional electrophoresis

6. Spectroscopic techniques II

*   

6.1. Methods of quantitative determination of proteins

6.2. Application of UV/VIS spectrophotometry to the kinetic analysis of enzymatic systems

6.3. Characterisation of enzymes; Determination of the type of inhibition

6.4. Methods of non-linear regression to determine kinetic constants of enzymatic reactions

7. Practical sessions

*   

7.1. Expression in E.coli of the cytosolic malate dehydrogenase from pig; Determination of the specific activity of the transformed cultures by UV/VIS spectrophotometric analysis and quantitative estimation of total protein; Determination of the molecular mass of the overexpressed protein by SDS-PAGE and general protein staining

7.2. Kinetics of reconstitution of the isoenzymes of lactate dehydrogenase; Native electrophoresis in polyacrylamide gels of the recombined isoforms; Protein-specific staining by means of enzymatic activity

7.3. Kinetic characterisation of the pyruvate-NADH-lactate dehydrogenase system; Determination of the type of reversible inhibition by oxamate and oxalate; Evaluation of their inhibition constants (Ki) and the IC50

7.4. Recombining expression and chromatographic purification of GFP (green fluorescent protein); Analysis by SDS-PAGE and general protein staining

7.5. Determination of blood Rh factor by PCR amplification of genomic DNA and electrophoretic analysis in agarose gels

7.6. Oligonucleotide site mutagenesis to reinstate the fluorescence of a mutated non-fluorescent GFP

7.7. Use of computer programs to analyse the sequence and structure of proteins and PCR primers used in practical sessions

* Unless the requirements enforced by health authorities demand a prioritisation or reduction of these contents.

 

 

Teaching methods and general organization

 

The subject Laboratory III includes a series of theoretical and practical sessions which allow students to learn and practice some common experimental techniques of biochemistry and molecular biology. Students distribute their time to plan and perform a series of experiments and to discuss the results, following the instructors’ indications and under their supervision.

With the aim of helping students’ work and optimise the resources, classroom and laboratory sessions follow these general rules:

— Class attendance to theoretical and practical sessions is mandatory.

— Lecturers explain in the classroom the theoretical bases of the experimental techniques that are subsequently put into practice in the laboratory.

— Students are distributed in groups of 20-24 participants.

— Within each group, students work in pairs to perform the assigned experiments.

— At the beginning of the course a detailed protocol of the practical sessions is provided to students, who must read, understand and solve any questions they may have before starting the corresponding experiment.

— Every student must write on his or her laboratory record book the procedures followed, any changes or experimental deviations from the proposed protocol, and the calculations and results obtained in the experiments.

— At the end of the practical sessions students share and discuss the results obtained by the different groups.

— A laboratory final report following the instructions provided must be presented in due time.

 

* The proposed teaching methodology may experience some modifications depending on the restrictions to face-to-face activities enforced by health authorities.

 

 

Official assessment of learning outcomes

 

Class and laboratory attendance are compulsory and continuous assessment comprises the following activities and weighting:

— Laboratory assessment: given the reduced number of students in each group, lecturers can monitor students’ attendance (which is compulsory), punctuality, interest, ability to work in group, dexterity in planning and performing the experiments. To pass the subject it is mandatory to pass this part, which represents 15% of the final grade.

— Laboratory report assessment: every pair of students must write a final laboratory report, following the indications of the lecturers. For every experiment performed in the laboratory, the final report must contain an introduction, a description of the experimental work, a results and discussion section and references. To pass the subject it is mandatory to pass this part, which represents 25% of the final grade.

— Final examination: it consists of a multiple-choice test plus some short-answer questions and/or practical exercises related to the theoretical or practical aspects of the topics discussed in class or in the laboratory. Students must not only be able to perform laboratory work but also understand the physical and chemical principles upon which the common experimental techniques in a Biochemistry laboratory are based. To pass the subject it is mandatory to pass this part, which represents 60% of the final grade.

 

Examination-based assessment

Given the experimental nature of the subject and since class and laboratory attendance is compulsory, single assessment is not available.

* Students’ assessment may experience some modifications depending on the restrictions to face-to-face activities enforced by health authorities.