IGEM:IMPERIAL/2009/M2/Assays/2.5

Background:

Fortunately, a visual analysis can be used to determine whether colanic acid is being produced by the chassis. This approach can be complimented with electron microscopy.

Reagents:

• LB Media Powder

• LB Agar Powder

• HSL(3OC6HSL)

• India Ink (colloidal carbon)

Equipment:

• Conical flask

• Foam stopper

• Autoclave

• Agar plates

• Strippette

• P1000 (Gilson)

• Incubator

• UV light source.

• Microscope

Wet Lab Protocol:

Day 1:

Prepare LB Agar Plates

• Measure out 7.4 grams of LB agar powder into a conical flask.

• Add 200 ml of H20 to the conical flask.

• Place foam stopper in top of conical flask and cover stopper in foil.

• Autoclave conical flask to sterilise media.

Preparation of LB Media (Starter Culture)

• Measure out 2.5 grams of LB media powder into a conical flask.

• Add 100 ml of H20 to the conical flask.

• Place foam stopper in top of conical flask and cover stopper in foil.

• Autoclave conical flask to sterilise media.

Day 2:

Pour LB Agar Plates

• Melt sterilised agar in the microwave (~2.5 mins, full power).

• Place conical flasks in a water bath (50 degrees centigrade) to prevent the agar from solidifying.

• After 20 minutes, add sufficient HSL to create a 1E-4 M solultion of HSL(3OC6HSL). Also ad an appropriate antibiotic if cells have a selection marker.

• Pour plates 8 plates.

• Allow to cool and place in cold room overnight.

Innoculate LB Media (Starter Culture)

• Using a sterile strippette, fill 8 falcon tubes each with 10ml of sterile LB media.

• Pick four different transformed colonies and replicate on 'replica plates' as well as transferring to four of the media-filled falcon tubes.

• Repeat the above step with four un-transformed colonies to serve as a control.

• Incubate the cells overnight at 37 degrees centigrade on a shaking incubator.

Day 3:

• Using a Gilson P1000, transfer 1ml from each falcon tube onto an appropriatly labelled agar plate.

• Keep the remaining starter cultures for Assay 2.6.

• Incubate plates overnight.

Day 4:

• Place agar plates under a UV light source. Green colouration indicates successful transformation with construct.

• Observe for visual signs of mucoidy.

• Stain cells with India Ink and observe under light microscope.

• View cells under electron microscope if available.

Above: Electron micrograph of colanic acid encapsulated cell.