Mobile genetic elements of bacteria 1400-215PLAZB
Laboratory part: I. Identification and analysis of bacterial plasmids: (1) Purification of plasmid DNA by CsCl-EtBr density gradient centrifugation; other methods of plasmid purification; visualization of mega-sized plasmids. (2) Construction of mini-replicons (defining of minimal and basic replicons); application of shuttle vectors for analyses of plasmid-derived replication and stabilization systems. (3) Plasmid vectors in genetic engineering: (a) different types of vectors; (b) isolation of DNA restriction fragments from agarose gels; (c) cloning of bacterial genes in different vectors (diverse procedures for selection of recombinant clones). (4) Introduction of plasmid DNA into bacterial cells: chemical transformation, electroporation, tri-parental conjugation (factors that influence plasmid maintenance in different hosts - host range, incompatibility and restriction barrier). II. Analysis of stabilization systems (TA; encoding toxin and antitoxin proteins) derived from different types of mobile genetic elements (plasmid, bacteriophage and conjugative transposon): (1) Analysis of genetic organization of addiction systems - isolation and electrophoresis of RNA, reverse transcription PCR (RT-PCR). (2) Identification of promoters and determination of their activity - ß-galactosidase activity assay. (3) Application of bacterial two-hybrid systems for detection of protein-protein (toxin-antitoxin) interactions. (4) Functional analyses of TA systems with application of inducible expression vectors: (a) analyses of the influence of toxin proteins on cells viability, (b) demonstration of the ability to reverse the toxic effect by the cognate antitoxins. III. Identification and characterization of transposable elements (TE) in bacteria: (1) Entrapment vectors as a tool for identification of TEs. (2) Determination of copy number and localization of TEs within the host genome by DNA-DNA hybridization (DIG-labeled probes). (3) Analyses of distribution of TEs in bacterial genomes by DNA-DNA hybridization (DOT-BLOT). IV. Bioinformatic analyses of the nucleotide sequences of TEs (programs: Artemis, BLAST, CloneManager, ORF Finder, etc.).
Type of course
Mode
Prerequisites (description)
Course coordinators
Learning outcomes
Having completed the course and the lab the student:
KNOWLEDGE
- Has up-to-date knowledge in the main areas of bacterial genetics including the scientific terminology concerning microbiology, genetics and genomics, the most recent researches, discoveries and applications of mobile genetic elements in biotechnology, medicine and molecular biology.
- Is familiar with the rules of research planning, up-to-date techniques of data collection and the use of various research tools, including a broad range of plasmid vectors.
- Knows how to: (i) independently plan and perform experiments in the field of genetic analyses of microorganisms, (ii) present the obtained results in a form appropriate for discussion, evaluation or publication (iii) the importance of experimental work in the bacterial genetics.
- Has the knowledge of the cellular and molecular basis for microorganism functions in light of the research on bacterial mobile genetic elements.
ABILITIES
- Uses broadly understood advanced molecular genetics techniques which make selection and targeted modification of microorganisms and mobile genetic elements possible.
- Has knowledge of a modern language (Polish or English) sufficient for using electronic resources and scientific literature devoted to broadly defined bacterial genetics.
- Is able to draw proper conclusions and interpret research results of molecular analyses based on the obtained data.
- Is able to plan easy experiments with the usage of mobile genetic elements (cloning, mutagenesis etc.).
- Is able to study on his/her own and focus on the study area.
SOCIAL AWARENESS
- Appreciates the significance of statistical and bioinformatic tools for reporting the results of experiments as well as processes occurring in nature.
- Is responsible for the research which has been assigned to him/her, for his/her own laboratory work and other people’s work.
- Is careful and critical when acquiring and interpreting knowledge in microbial genetics and its practical applications.
- Is responsible for the assessment of the risks associated with the research techniques used and for ensuring safe working conditions.
- Understands the need for informing the public about latest achievements in biology of microorganisms and mobile genetic elements and is able to pass this knowledge in an intelligible way.
Assessment criteria
Laboratory classes are passed when the student:
1) attended at least 85% of the classes;
2) was working during the classes in the way enabling positive evaluation of his knowledge, abilities and social awareness gained during the classes (as described in learning outcomes).
Detailed conditions:
- student’s activity during the classes;
- passing the descriptive exam (gaining at least 51% of points)
The final score from the subject is the final exam score.
The conditions obligatory for passing the final exam:
1) Passing the exam from the laboratory part.
2) Passing the final exam in a form of a multiple choice test (20) and a descriptive exam (10), i.e. gaining at least 51% of points.
Practical placement
No.
Bibliography
1. Biologia Molekularna Bakterii. Baj J., Markiewicz Z. (ed.) PWN, 2015
2. Plasmid Biology. Funnel B.E., Philips G.J. (ed.) 2003
3. The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread. Thomas C.M. (ed.). 2000
4. Mobile DNA III. Craig N.L., Chandler M., Gellert M., Lambowitz A.M., Rice P.A., Sandmeyer S.B. (ed.) 2015
5. Bacterial Integrative Mobile Genetic Elements. Roberts A.P. and Mullany P. (ed.) 2013
6. Mikrobiologia. Baj J. (ed.) PWN, 2018
7. Arnold BJ, Huang IT, Hanage WP. Horizontal gene transfer and adaptive evolution in bacteria. Nat Rev Microbiol. 2022 Apr;20(4):206-218. doi: 10.1038/s41579-021-00650-4.
8. Cambray G, Guerout AM, Mazel D. Integrons. Annu Rev Genet. 2010;44:141-66. doi: 10.1146/annurev-genet-102209-163504.
9. diCenzo GC, Finan TM. The divided bacterial genome: structure, function, and evolution. Microbiol Mol Biol Rev. 2017 Aug 9;81(3):e00019-17. doi: 10.1128/MMBR.00019-17.
10. Haudiquet M, de Sousa JM, Touchon M, Rocha EPC. Selfish, promiscuous and sometimes useful: how mobile genetic elements drive horizontal gene transfer in microbial populations. Philos Trans R Soc Lond B Biol Sci. 2022 Oct 10;377(1861):20210234. doi: 10.1098/rstb.2021.0234.
11. Horne T, Orr VT, Hall JP. How do interactions between mobile genetic elements affect horizontal gene transfer? Curr Opin Microbiol. 2023 Jun;73:102282. doi: 10.1016/j.mib.2023.102282.
12. Jurėnas D, Fraikin N, Goormaghtigh F, Van Melderen L. Biology and evolution of bacterial toxin-antitoxin systems. Nat Rev Microbiol. 2022 Jun;20(6):335-350. doi: 10.1038/s41579-021-00661-1.
13. Lang AS, Buchan A, Burrus V. Interactions and evolutionary relationships among bacterial mobile genetic elements. Nat Rev Microbiol. 2025 Mar 11. doi: 10.1038/s41579-025-01157-y.
14. Lipszyc A, Szuplewska M, Bartosik D. How do transposable elements activate expression of transcriptionally silent antibiotic resistance genes? Int J Mol Sci. 2022 Jul 22;23(15):8063. doi: 10.3390/ijms23158063.
15. M, Firth N, Jensen SO. Mobile genetic elements associated with antimicrobial resistance. Clin Microbiol Rev. 2018 Aug 1;31(4):e00088-17. doi: 10.1128/CMR.00088-17.
16. Rodríguez-Beltrán J, DelaFuente J, León-Sampedro R, MacLean RC, San Millán Á. Beyond horizontal gene transfer: the role of plasmids in bacterial evolution. Nat Rev Microbiol. 2021 Jun;19(6):347-359. doi: 10.1038/s41579-020-00497-1.
17. Tokuda M, Shintani M. Microbial evolution through horizontal gene transfer by mobile genetic elements. Microb Biotechnol. 2024 Jan;17(1):e14408. doi: 10.1111/1751-7915.14408.
18. Vos M, Buckling A, Kuijper B, Eyre-Walker A, Bontemps C, Leblond P, Dimitriu T. Why do mobile genetic elements transfer DNA of their hosts? Trends Genet. 2024 Nov;40(11):927-938. doi: 10.1016/j.tig.2024.07.008.
19. Weisberg AJ, Chang JH. Mobile genetic element flexibility as an underlying principle to bacterial evolution. Annu Rev Microbiol. 2023 Sep 15;77:603-624. doi: 10.1146/annurev-micro-032521-022006.
20. Weltzer ML, Wall D. Social diversification driven by mobile genetic elements. Genes (Basel). 2023 Mar 4;14(3):648. doi: 10.3390/genes14030648.
Notes
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Term 2024Z:
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Term 2025Z:
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