Team:Newcastle/Project/plants

From 2013.igem.org

(Difference between revisions)
(→Introducing and detecting L-forms in Plants)
Line 1: Line 1:
{{Team:Newcastle}}
{{Team:Newcastle}}
==Introducing and detecting L-forms in Plants==
==Introducing and detecting L-forms in Plants==
-
===Detecting L-forms in Plants Using gusA Reporter Gene===
+
L-forms in plants
-
 
+
BioBrick: GUS reporter BioBrick
-
<b>Transformation ''Bacillus subtilis'' to contain gusA</b>
+
Purpose of BioBrick: To act as a reporter system to observe the localisation of Bacillus subtilis in plants.
-
 
+
Introduction to Project: It is thought that L-forms may exist in plants in a symbiotic relationship with the plant providing a suitable environment for the osmolarity sensitive bacteria. In return, L-forms can confer benefits to their host including reducing the rate of fungal infection. L-forms have ethical benefits, as they will lyse upon leaving their host plants. It may be possible to create L-forms which produce and supply nutrients to the plant they reside in. This will be especially useful if the L-forms host is unable to synthesise the nutrients provided by the bacteria. Therefore, in the future we could use L-forms to support crop growth in nutrient-lacking soil with the knowledge that the L-forms cannot escape into the environment.
-
1)    Transform L-forms of ''B. subtilis'' NCIMB8054.
+
In order to prove that L-forms can grow in plants, we aim to transform B. subtillis strain LR2 with the gusA reporter gene coding for β-glucuronidase. We will remove gusA BioBrick from its plasmid backbone and attach it to another plasmid containg AprE for homologous recombination and kanamycin resistance for selection. We can then transform B. subtillis with the new transformation vector. The bacteria that have taken up the plasmid can then be selected for via kanamycin resistance. A transformation vector is used rather than a normal plasmid so the gusA is integrated through homologous recombination into the bacteria’s chromosome, securing the expressing of the gusA.
-
 
+
The β-glucuronidase produced by the L-forms in plants will cleave the colourless substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) to produce a blue product allowing the L-form distribution in plant tissue to be observed. Once we have introduced L-forms into plants, it may be possible to see which plant tissues L-forms colonize at a high concentration, and so infer which areas of the plant best suit L-form growth.
-
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===
===Alternative Methods Considered===

Revision as of 12:36, 9 July 2013

 
X
 
IGEM Home Newcastle University

Introducing and detecting L-forms in Plants

L-forms in plants BioBrick: GUS reporter BioBrick Purpose of BioBrick: To act as a reporter system to observe the localisation of Bacillus subtilis in plants. Introduction to Project: It is thought that L-forms may exist in plants in a symbiotic relationship with the plant providing a suitable environment for the osmolarity sensitive bacteria. In return, L-forms can confer benefits to their host including reducing the rate of fungal infection. L-forms have ethical benefits, as they will lyse upon leaving their host plants. It may be possible to create L-forms which produce and supply nutrients to the plant they reside in. This will be especially useful if the L-forms host is unable to synthesise the nutrients provided by the bacteria. Therefore, in the future we could use L-forms to support crop growth in nutrient-lacking soil with the knowledge that the L-forms cannot escape into the environment. In order to prove that L-forms can grow in plants, we aim to transform B. subtillis strain LR2 with the gusA reporter gene coding for β-glucuronidase. We will remove gusA BioBrick from its plasmid backbone and attach it to another plasmid containg AprE for homologous recombination and kanamycin resistance for selection. We can then transform B. subtillis with the new transformation vector. The bacteria that have taken up the plasmid can then be selected for via kanamycin resistance. A transformation vector is used rather than a normal plasmid so the gusA is integrated through homologous recombination into the bacteria’s chromosome, securing the expressing of the gusA. The β-glucuronidase produced by the L-forms in plants will cleave the colourless substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) to produce a blue product allowing the L-form distribution in plant tissue to be observed. Once we have introduced L-forms into plants, it may be possible to see which plant tissues L-forms colonize at a high concentration, and so infer which areas of the plant best suit L-form growth.

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.

Retrieved from "http://2013.igem.org/Team:Newcastle/Project/plants"