IGEM:IMPERIAL/2009/M2/Assays/2.5

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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.


CA.JPG Above: Electron micrograph of colanic acid encapsulated cell.