Molecular Biology

obligatory courses

Biochemistry 1400-113BCH
Genetics and Genetic Engineering 1400-114GEN

Students are obliged to complete the 1st year of the first level studies (Bachelor's) before starting the subject

Classroom

Selected genomes and their evolution. Methods for genome sequencing. DNA microarrays. DNA replication. Mechanisms of prokaryotic gene expression regulation at the level of transcription initiation and post-transcriptionally. Global regulation of gene expression in bacteria (regulons, quorum sensing). Chromatin and the architecture of the cell nucleus. Regulation of transcription in eukaryotes. The RNA interference. Epigenetic mechanisms. Translation in eukaryotes. Amino acids and the polypeptide chain. Protein folding, structure, and post-translational modifications.

Laboratory classes are divided into three blocks run by employees of the Institute of Biochemistry, the Institute of Experimental Biology and Plant Biotechnology, and the Institute of Microbiology.There are four meetings within each part. Classes cover methods of DNA cloning and manipulation, recombinant proteins isolation, methods of plant molecular biology and basics of DNA sequence analysis and databases.

Lecture.

DNA structure. DNA replication in eukaryotic organisms. Details of Okazaki fragment structure and role in DNA replication and issues regarding its termination. The role and structure of telomere. DNA damage types and their repair mechanisms. Non-homologous end joining and homologous recombination.

Tools in molecular biology and DNA sequencing. Chemical synthesis of oligonucleotides and their application in PCR. Enzymes utilised in molecular biology laboratory procedures. Methods for genome sequencing: Sanger’s method and next generation sequencing. The history of human genome sequencing. DNA data storage.

Genome structure, its elements and evolution. The first sequenced genomes and their characteristics. Hypotheses regarding the evolutionary origin of genes. Key structural elements of genes. Genome organisation. Transposable elements, their structures and mechanisms of transposition.

Examples of different regulatory mechanisms of gene expression in prokaryotic organisms at the level of initiation and termination of transcription (e.g. Lac operon, arabinose operon, tryptophan operon. attenuation). Expression of prokaryotic genes: regulatory mechanisms. Regulation at the following levels (i) post-transriptional level with the participation of proteins and regulatory RNA (including riboregulation, riboswitches), (ii) translation and (iii) post-translational level. Operon, regulon, modulon. Global regulation at the cellular (including a two-component regulatory system) and the entire population level (including quorum sensing). DNA replication and recombination in bacteria.

Regulation of gene expression in eukaryotes, particularly in relation to the hierarchical organisation of chromatin and the spatial architecture of the cell nucleus. Liquid-liquid phase separation (LLPS) phenomenon, which enables the formation of transcriptional condensates containing RNA polymerase II and regulatory factors. Transcription, including initiation, elongation and termination carried out by RNA polymerase II. The role of enhancers, superenhancers and transcription factors in fine-tuning gene regulation. Maturation of mRNAs such as splicing, polyadenylation and export. Small RNAs (miRNAs, siRNAs, piRNAs) and their involvement in gene regulation and the maintenance of genome integrity.

Eukaryotic translation. Amino acids and polypeptide chain. Primary, secondary, tertiary and quaternary protein structure. Protein folding. Chaperones, chaperonins. Posttranslational modifications of proteins.

Laboratory.

Restriction endonucleases and enzymes used to modify DNA. DNA recombination and cloning. Methods applied to introduce DNA to bacterial cells. Competent cells and bacterial transformation. Plasmid vectors. Antibiotics applied in molecular biology. Isolation of plasmid DNA, DNA electrophoresis, estimation of its purity and quality. Application of Escherichia coli lac operon in DNA cloning. Methods applied to the selection of recombinant DNA, alfa-complementation. Catabolic repression.

Purification of the recombinant enzymatic protein expressed in E. coli cells by the means of immobilized-metal affinity chromatography (IMAC). Evaluation of the quality of protein preparations, what includes: i) evaluation of catalytic activity in the protein preparation, ii) analysis of protein purity by SDS polyacrylamide gel electrophoresis (SDS PAGE) (various gel staining techniques, Coomassie Brilliant Blue staining, silver staining and staining with fluorescent dye, will be compared), iii) immunological identification of the protein by Western blotting (secondary antibodies conjugated with alkaline phosphatase or horseradish peroxidase will be used and various detection methods, including enhanced chemiluminescence, will be applied).DNA isolation from plant cells.

Transgenic plants as examples of genetically modified organisms - generation, application and analysis. Transformation of tobacco plants - leaf infiltration with the Agrobacterium tumefaciens bacterial culture carrying the genetic construct with the marker gene.

Reporter genes, like green fluorescent protein (GFP), luciferase, glucouronidase (GUS), etc, often used to indicate of whether a certain gene has been taken up by or expressed in the plant . The methods of testing protein interactions, such as yeast two-hybrid system, FRET (Föster Resonance Energy Transfer), and BiFC (Bimolecular fluorescence complementation) will also be discussed and presented. The transient transformation of plants will be performed and the introduced gene expression will be analysed by observing the green fluorescence protein under the fluorescence microscope. The DNA constructs used during the experiment contain genes encoding GFP translational fusions with proteins from various parts of the cell. This will allow the localization of these proteins to be analyzed. Observations of plants expressing the glucuronidase gene under the control of various tissue-specific promoters will be carried out.

The course will discuss the real-time PCR technique, principles of operation, including specific and non-specific detection of nucleic acids, and issues related to experiment planning, such as the selection of reference genes and the design of oligonucleotides. Variants of the method and possible applications will be presented.

Molecular biology methods used in analyses of Arabidopsis mutants and transgenic lines. Plant genetics - forward and reverse genetics approaches for Arabidopssi thaliana model; mutagenesis and forward genetic screen of Arabidopsis mutants; mapping of point and insertional mutations, PCR analysis of polymorphic sequences in two Arabidopsis ecotypes.

Materials provided by the teaching team.

Podstawy biologii molekularnej. L.A. Allison, Wyd. UW, 2009

Biologia molekularna. Krótkie wykłady. P. Turner i in., PWN, 2005 or later

Techniki laboratoryjne w biologii molekularnej. A. Lewandowska-Ronnegren, MedPharm, 2017

Podstawy biologii komórki. B. Alberts i in., PWN, 2019 or later

Biologia molekularna bakterii. J. Baj, Z. Markiewicz, PWN, 2015

Mikrobiologia. J. Baj, PWN, 2018

Biochemia. J.M. Berg, J.L. Tymoczko, L. Stryer. PWN, 2005 or later

Biochemia Harpera, V.W. Rodwell i in., PZWL, 2004 or later

Genomy. T.A. Brown, PWN, 2009 or later

Genes IX. B.Lewin, Jones and Bartlett Publ., 2007 or later

Brock Biology of Microorganisms. M.T. Madigan, J.M. Martinko

Introduction to Protein Structure. C. Branden, J. Tooze, Garland Publ., 1999 (supplementary, for those interested in the subject)

Introduction to Protein Architecture, A.M. Lesk, Oxford University Press, 2001 (supplementary textbook, for those interested in the subject)

Having completed the course and the lab the student:

KNOWLEDGE:

1. has a basic knowledge of molecular biology. Knows and understands the molecular basis of functioning of prokaryotic and eukaryotic cells (K_W01 S1-BT, K_W12 S1-BI).

2. can identify the most important scientific discoveries in the history of molecular biology (K_W02 S1-BT).

3. knows the basic techniques and tools used in the study of natural phenomena in molecular biology (K_W04 S1-BT, K_W14 S1-BI).

4. knows the basics of designing and performing genetic modifications on biological material (K_W04 S1-BT).

5. knows the basics of information technology and uses computer tools to extract information from selected databases (K_W08 S1-BT, K_W16 S1-BI).

6. knows the principles of safety and hygiene in molecular biology laboratory (K_W09 S1-BT, K_W17 S1 BI).

SKILLS

1. is able to apply basic molecular biology techniques (K_U01 S1-BT, K_U01 S1-BI).

2. is able to use basic databases of scientific articles as well as DNA and protein sequences (K_U03 S1-BT, K_U08 S1-BI).

3. performs basic research experiments in molecular biology under the supervision (K_U04 Bt1). (K_U04 S1-BT, K_U05 S1-BI).

4. demonstrates the ability to reason correctly on the basis of experimental data (K_U06 S1-BT, K_U03 S1-BI).

SOCIAL COMPETENCES:

1. Demonstrates responsibility for their own safety and that of others (K_K03 S1-BT, K_K05 S1-BI).

2. Demonstrates responsibility for the equipment entrusted to them in the molecular biology laboratory.

3. Is prepared to work effectively in a team (K_K04 S1-BT, K_K07 S1-BI).

In order to complete the laboratory student is obliged to: (i) attend at least 85% of classes; (ii) work during the laboratory classes in a way that allows positively assess the knowledge, skills and social competence, which were obtained in the course of the classes (described in the syllabus as a subject educational outcomes); (iii) pass the final test.

Detailed conditions: (i) attendance and active participation in the classes; (ii) fulfillment of tasks assigned for self preparation (iii) passing the written test, including both open and close-ended questions. Duration of the test: 90 minutes. To pass the test, one should get more than half of the points.

In order to complete the whole course student is obliged to pass the final exam, for which one is admitted on the basis of assessment of laboratory classes. Form of the exam: written (single-choice test, multiple choice test, open-ended questions, essay questions). The condition for passing it is to obtain 60% of the possible points.

No.