Bacterial Transformation The bacterium Bacterial chromosome Plasmids Cytoplasm

Bacterial Transformation

The bacterium Bacterial chromosome Plasmids Cytoplasm Ribosomes Cell wall Cell membranes

The plasmid GFP gene This gene encodes green fluorescent protein, which glows in UV light

Aequorea victoria

Green fluorescent protein

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

v Wipe the bench surface with 1 % bleach before and at the end of the procedure v Wash hands before and at the end v 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

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

Plasmid DNA in buffer

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

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

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

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

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

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.

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.

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.

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

BIOHAZARD TRANSFORMED CELLS MUST BE DESTROYED AFTER USE

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

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