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Bacterial Transformation and GFP stories

Bacterial Transformation and GFP stories

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Bacterial Transformation and GFP stories

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  1. Bacterial Transformation and GFP stories Kate Andrews Lorraine Bruce Marjorie Smith

  2. The bacterium Bacterial chromosome Plasmids Cytoplasm Ribosomes Cell wall Cell membranes

  3. The plasmid Origin of replication this is needed so that the plasmid can be copied, using the bacterium’s DNA copying machinery Kanamycin resistance gene -the enzyme encoded by this gene stops the antibiotic kanamycin from working This gene encodes green fluorescent protein, which glows in UV light GFP gene

  4. Aequorea victoria

  5. Green fluorescent protein

  6. Someone trained to level 3 • media preparation and disposal • sub-culturing and storage of cultures • preparation and maintenance of spills kit • sterilisation of equipment http://www.sserc.org.uk/index.php/health-safety/health-a-safety-home140

  7. Good microbiological laboratory practice (GMLP) • Regard all micro-organisms as potential pathogens • Cover exposed cuts and abrasions with waterproof • dressings • Wash hands before and after practical work • Laboratory windows and doors should be closed

  8. Wipe the bench surface with 1 % bleach before and • at the end of the procedure • Wash hands before and at the end • Work within a 20 cm radius of a lit bunsen flame (blue) • to create an up-flow of warm air which will carry away • any potentially contaminating organisms

  9. Label plates carefully (plates on underside) • Handle, pipettes , loops and spreaders carefully – • dispose of these into Virkon™ • Don’t put lids of plate on bench….. • Inoculated plates should be sealed diametrically • with small pieces of sticky tape

  10. Plasmid DNA in buffer

  11. Label the Petri dish you are going to inoculate: • Small, neat writing • Edge of plate • Your initials, the date and the name of the bacterium

  12. Use a sterile loop to pick up one, or two colonies of bacteria from the stock plate

  13. Put the loop of bacteria into the ice-cold plasmid DNA solution... Hold the tube almost horizontally Place the contaminated loop in the discard jar

  14. Close the tube tightly and put on ice for 15 minutes. This allows the bacteria to take up the plasmid DNA.

  15. Aims: • ...key technique used in genetic modification • ...basic microbiological techniques • ...context for discussion of some of the ethical, social and safety issues associated with genetic modification • ......plan and carry out (necessarily limited) open-ended practical investigations

  16. Curriculum links Human Cells Metabolism and Survival Control of metabolic pathways (presence or absence of particular enzymes) and the regulation of the rate of reaction of key enzymes within the pathway Enzyme induction experiments such as ONPG and lactose metabolism in E. coli and PGlo experiments. Regulation can be controlled by intra- and extra cellular signal molecules.

  17. Experimental design...... Thinking about ‘controls’........ -pGLO LB/kanamycin +pGLO LB/kanamycin -pGLO LB +pGLO LB/amp/ara Bacteria with plasmid can grow. They have antibiotic resistance. IPTG has switched on GFP protein expression No plasmid but nothing to stop bacteria growing. No plasmid therefore bacteria are killed by ampicillin. Bacteria with plasmid can grow. They have antibiotic resistance. No IPTG therefore no GFP expression

  18. Use a sterile pipette to place 3 drops of bacterial suspension onto the centre of the your Petri dish. Place the contaminated pipette and the opened tube of bacteria into the discard jar.

  19. Use a sterile spreader to coat the surface of the agar with the bacterial suspension. Rotate the plate as you do this, so that the culture is spread evenly over the plate. Place the contaminated spreader in the discard jar. Seal the Petri dish with adhesive tape – 2 small pieces opposite sides.

  20. Petri dishes should be incubated upside down at 300C Clean the work area with 1% bleach. Wash your hands.

  21. BIOHAZARD TRANSFORMED CELLS MUST BE DESTROYED AFTER USE

  22. After about 24 hours, examine the bacteria under ultraviolet light.

  23. The assignment • investigation stage: • select an appropriate biology topic… • investigate/research the topic, focusing on applications and • impact on society/the environment • process the information/data collected • controlled assessment stage: • knowledge and understanding • application • balanced evaluation • reasoned conclusion

  24. http://www.plantsci.cam.ac.uk/Haseloff/imaging/cell_images/awards/awards.htmlhttp://www.plantsci.cam.ac.uk/Haseloff/imaging/cell_images/awards/awards.html

  25. GFP stories