Contents 1 L-forms in Plants 1.1 BioBrick 1.2 Purpose of BioBrick 1.2.1 Method 1.3 Alternative Methods Considered 1.4 References

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.

Method

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.