Introducing and Detecting L-forms in Plants

Introducing and detecting L-forms in Plants

Detecting L-forms in Plants Using gusA Reporter Gene

Transformation B.subtilis to contain gusA

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 gusA 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 deionised 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 gusA 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 L-phase medium (which is designed for the growth of L-forms) and nutrient agar and incubate (also repeat using 100ul of original bacterial suspension on each of agar).

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

GusA Reporter Gene

Gus A reporter gene encodes beta-glucornide (GUS) an enzyme in Escherichia coli. BioBrick Part: BBa_K330002 [http://partsregistry.org/Part:BBa_K330002:Experience]

Detecting L-forms in Plants Using Red Flourescent Protein

Transformation B.subtilis to contain RFP gene

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 RFP 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 distillled water to lyse extracellular L-forms.

6) Incubate plants.

Show RFP 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 RFP 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 L-phase medium and nutrient agar and incubate (also repeat using 100ul of original bacterial suspension for each agar).

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

References

Tsomlexoglou, E., Daulagala, P.W.H.K.P., Gooday, G.W., Glover, L.A., Seddon, B. and Allan, E.J. (2003) 'Molecular detection and β-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings', Journal of Applied Microbiology, 95(2), pp. 218-224.

Ferguson, C.M.J., Booth, N.A. and Allan, E.J. (2000) 'An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry', Letters in Applied Microbiology, 31(5), pp. 390-394.