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.