Contents 1 Project Overview 1.1 Genome Shuffling 1.2 Introduction and Detection of Naked Bacteria in Plants 1.3 Shape Shifting 1.4 Modelling 1.5 References

Project Overview

We have created a BioBrick which makes the model Gram positive bacteria Bacillus subtilis lose or regain its cell wall, on our demand, while still allowing it to grow and divide. B. subtilis without a cell wall is better in many different ways and should be used as a new chassis for use in Synthetic Biology.

All you need to start using an L-form chassis is a culture of Bacillus subtilis, our L-form switch BioBrick and a set of instructions from us. Bacteria which have lost their cell wall which are still able to grow and propagate are called L-forms, or as we prefer to call them, naked bacteria. There are loads of things that you can do with naked bacteria, we explored a few of them:

(One of the best facts about naked bacteria is that they are osmotically sensitive, meaning that they will lyse if they escape into the environment. This means that they can be used in non-contained environments.)

Genome Shuffling

Fusion of bacteria is made significantly easier without a cell wall. This forces the fusants to reproduce sexually, where their genomes recombine, and this can be used in directed evolution. We have shown L-forms fusing, and the recombination of their genomes.

Introduction and Detection of Naked Bacteria in Plants

L-forms have been shown to form symbiosis in plants. We’ve shown that the naked bacteria that we created using our switch BioBrick also form these associations. Plants with naked bacteria show increased resistance to fungus and they could be used to deliver useful compounds to the plant. This could give better plant yields, more nutritious plants and reduce the need for spraying of fertiliser, pesticides or other compounds.

Shape Shifting

The loss of the cell wall leaves L-forms protected by only a cell membrane. The plasma membrane of l-forms is quite fluid. The advantage of this is that these cells would be able to adapt to shapes of various cracks and cavities, or will be able to "squeeze through" tiny channels and deliver cargo to hard-to reach targets.

We were planning to test this hypothesis by injecting the naked bacteria into specially designed microfluidics chambers and observing their behaviour under the microscope. However due to time and logistics constraints we were unable to do it. For more information please visit shape shifting page

Modelling

We understand the importance of modelling in synthetic biology and have attempted to model as much as we were able. We constructed a model of membrane behaviour of L-forms, which included stress conditions, cell growth and boundaries provided by the chamber walls. We have modelled the biophysical mechanism of the L-form cell membrane fusion. We also modelled what effect the BioBricks we developed would have on B. subtilis.

This isn’t a finite list of what can be done with naked bacteria, there’s loads more that can be done! L-forms are currently used to discover novel antibiotics which don’t act on the cell wall. L-forms can teach us a great deal about how bacterial life has evolved, through acting as a model for a cell wall-less bacterial progenitor, and through being able to test the ease of induction of endosymbiosis in cell wall-less organisms (Mercier et al. 2013).

L-form bacteria can be used in any process which protoplasts are currently used for. Protoplasts are bacteria which have been chemically induced to lose their cell wall. They cannot however grow or divide (as L-forms can) and are not classified as being alive. L-forms can be used to transform bacteria which are recalcitrant to transformation (Chang and Cohen 1979).

References

Walker R, Ferguson CMJ, Booth NA and Allan EJ (2002) The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of ‘Botrytis cinerea. Letters in Applied Microbiology, 34, 42-45.

Chang S and Cohen SN (1979)High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molecular Genetics & Genomics, 168, 111–115.

Mercier R, Kawai Y and Errington J. (2013) Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation, Cell, 152, 997–1007.