"

From 2013.igem.org

Brief Overview of Detecting L-forms in Plants using Gus Reporter Gene

Transformation B.subtilis to contain gus A

1) Transform L-forms of B. subtilis NCIMB8054

2) Transformed L-forms will be selected by antibiotic resistance

3) Conduct southern hybridization to confirm the integration of the gus A gene

Production of L-form containing Plants

1) Wash seeds.

2) Grow seeds in petri dishes.

3) Incubate until radicals had just emerge.

4) Wash seeds with transformed L-forms.

5) Wash plants with distil water to lyse extracellular L-forms.

6) Incubate plants.

Visualisation of L-forms in plants

1) Heat seeds in a vacuum oven with gus staining solution.

2) Incubate at 37 degrees.

3) When glucuronidase activity appears, fix plants with formaldehyde.

Show gus gene is only present in transformed L-forms

1) Extract DNA from transformed L-form B. subtilis, L-form control and non L-form B.subtilis.

2) PCR: Use primers specific for gus A gene in a PCR to show gus A gene is present in transformed L-forms only.

Re-isolation of L-forms from seeds

1) Wash seeds treated with L-form bacteria or mannitol control with distilled water to remove any L-forms on the plant surface.

2) Place seeds in in mannitol solution.

3) Macerate seeds with pestle.

4) Plate out 100ul of suspension onto LPM and nutrient agar and incubate (also use 100ul of original bacterial suspension on each of these agar).

5) Look for signs of life, L-form or otherwise.