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

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

4. 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

5. Aequorea victoria

6. Green fluorescent protein

8. Someone trained to level 3 media preparation and disposal
sub-culturing and storage of cultures
preparation and maintenance of spills kit
sterilisation of equipment

9. 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

10. at the end of the procedure
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

11. Handle, pipettes , loops and spreaders carefully –
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

12. Plasmid DNA in buffer

13. 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

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

15. Place the contaminated loop in the discard jar
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

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

17. 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

18. 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.

19. Thinking about ‘controls’........
Experimental design......
Thinking about ‘controls’
-pGLO LB
-pGLO
LB/kanamycin
+pGLO
LB/kanamycin
+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

20. 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.

21. 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.

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

23. BIOHAZARD
TRANSFORMED CELLS MUST BE DESTROYED AFTER USE

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

26. 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

27. http://www. plantsci. cam. ac

28. GFP stories