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 (RFP)

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.

Alternative Methods Considered

Enzyme-linked immunosorbent assay (ELISA)

We considered producing an ELISA to detect L-forms. One method would be to generate antibodies against a L-form specific antigen. Alternatively we considered introducing a novel gene into L-forms to result in a novel antigen being produced for us to generate antibodies against. However this would have involved using animals to generate polyclonal antibodies. We decided this raised ethical issues and was too time consuming to complete in our ten week placement.

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.