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Introducing and Detecting L-forms in Plants
From 2013.igem.org
Contents |
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.
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.
