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Introducing and Detecting L-forms in Plants
From 2013.igem.org
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| - | =Brief Overview of Detecting L-forms in Plants Using | + | =Brief Overview of Detecting L-forms in Plants Using gusA Reporter Gene= |
| - | '''Transformation B.subtilis to contain | + | '''Transformation B.subtilis to contain gusA''' |
1) Transform L-forms of B. subtilis NCIMB8054 | 1) Transform L-forms of B. subtilis NCIMB8054 | ||
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2) Transformed L-forms will be selected by antibiotic resistance | 2) Transformed L-forms will be selected by antibiotic resistance | ||
| - | 3) Conduct southern hybridization to confirm the integration of the | + | 3) Conduct southern hybridization to confirm the integration of the gusA gene |
'''Production of L-form containing Plants''' | '''Production of L-form containing Plants''' | ||
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'''Visualisation of L-forms in plants''' | '''Visualisation of L-forms in plants''' | ||
| - | 1) Heat seeds in a vacuum oven with | + | 1) Heat seeds in a vacuum oven with GUS staining solution. |
2) Incubate at 37 degrees. | 2) Incubate at 37 degrees. | ||
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1) Extract DNA from transformed L-form B. subtilis, L-form control and non L-form B.subtilis. | 1) Extract DNA from transformed L-form B. subtilis, L-form control and non L-form B.subtilis. | ||
| - | 2) PCR: Use primers specific for | + | 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''' | '''Re-isolation of L-forms from seeds''' | ||
Revision as of 17:01, 12 June 2013
Brief Overview of 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 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 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 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.
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]
