Gene Transfer Lecture Outline PDF

GENE TRANSFER NURUL ASMA ABDULLAH, ASSOC. PROF., PH.D PRESENTATION TITLE LECTURE OUTLINE GENERAL OUTCOME: Understanding methods of gene transfer in bacterial systems...

GENE TRANSFER NURUL ASMA ABDULLAH, ASSOC. PROF., PH.D PRESENTATION TITLE LECTURE OUTLINE GENERAL OUTCOME: Understanding methods of gene transfer in bacterial systems SPECIFIC OUTCOME: To describe the basics of transformation, conjugation and transfection in bacterial systems 2 PRESENTATION TITLE GENETIC TRANSFER Transfer of genetic information between individuals is achieved by two mechanisms:  Vertical, from parent to siblings  Horizontal between individuals of the same or different species 3 4 PRESENTATION TITLE PRESENTATION TITLE VERTICAL GENE TRANSFER  Vertical gene transfer is referred to as the transfer of genetic information, including any genetic mutations, from a parent to their offspring.  It could happen by artificial means, asexual reproduction, or sexual reproduction.  This procedure is a typical mechanism that occurs in groups of related species. 5  HORIZONTAL PRESENTATION TITLE GENE TRANSFER  Is a process in which an organism transfers genetic material to another organism that is not its offspring  In horizontal gene transfer, newly acquired DNA is incorporated into the genome of the recipient through either recombination or insertion  Can cause large-scale changes in a bacterial genome 6  MECHANISM OF HORIZONTAL GENE TRANSFER PRESENTATION TITLE TRANSFORMATION CONJUGATION TRANSDUCTION 7  TRANSFORMATION PRESENTATION TITLE  A process in which bacteria take up free DNA from their surroundings.  In nature, free DNA typically comes from other lysed bacterial cells.  In the laboratory, free DNA in the form of recombinant plasmids is introduced to the cell’s surroundings.  Some bacteria, such as Bacillus spp., are naturally competent, meaning they are able to take up foreign DNA.  However, not all bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory 8  CONJUGATION PRESENTATION TITLE  Conjugation is the process by which one bacterium transfers genetic material to another through direct contact.  During conjugation, one bacterium serves as the donor of the genetic material, and the other serves as the recipient.  The donor bacterium carries a DNA sequence called the fertility factor, or F-factor.  Conjugation occurs in four (4) steps: 9 https://youtu.be/7Q9Eg1GV6WI?si=LlY9j98XoihwF-F6  TRANSDUCTION PRESENTATION TITLE  Transduction is the process by which a virus transfers genetic material from one bacterium to another  Viruses called bacteriophages are able to infect bacterial cells and use them as hosts to make more viruses  Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell’s genome 10 https://youtu.be/txSq-7BchUQ?si=i9rZ8pLhLlHia9hp  GENE TRANSFER METHOD PRESENTATION TITLE 11  ELECTROPORATION PRESENTATION TITLE A pulse of high voltage is applied to cells or tissues which makes transient pores in the plasma membrane through which uptake of foreign DNA occurs. 12 PRESENTATION TITLE Bacterial transformation using electrocompetent cells and electroporation.  Electroporation involves using an electroporator to expose competent cells and DNA to a brief pulse of a high-voltage electric field.  This treatment is to induce transient pores in cell membranes, which permit DNA entry into the cells  Typically, electroporation of bacteria utilizes 0.1 cm cuvettes (20–80 µL volume) and requires a field strength of >15 kV/cm. 13  Heat Shock Transformation PRESENTATION TITLE Using Calcium Chloride  E. coli is the most common bacterial species used in the transformation step of a cloning workflow.  Since the natural competency of E. coli is very low or even nonexistent, the cells need to be made competent for transformation by heat shock or by electroporation.  Competent cells are chemically prepared by incubating the cells in calcium chloride (CaCl2) to make the cell membrane more permeable 14 PRESENTATION TITLE CHEMICAL TRANSFORMATION  Heat shock is performed at 37– 42°C for 25–45 seconds as appropriate for the bacterial strain and DNA used.  For smaller volumes of cells in smaller tubes, the heat-shock interval, which depends on the surface-to-volume ratio of the cell suspension, should be reduced.  Heat-shocked cells are then returned to ice for ≥2 minutes before the next step 15  APPLICATIONS OF GENE TRANSFER PRESENTATION TITLE 1. Production of recombinant antibodies 2. Production of vaccines 3. Gene therapy 4. Production of transgenic animals 5. Gene discovery 6. Production of new improved crops 7. Production of pesticides/herbicides 8. Genetically modified organism 16 THANK YOU [email protected]