Project

Overview

We aim to investigate L-forms of the model Gram positive bacteria, Bacillus subtilis. L-forms are bacterial cells that are able to propagate and grow without a cell wall in contrast to protoplasts. We propose that L-forms can be used as a novel chassis for synthetic biology.

Furthermore, we are exploring some of the interesting applications that bacteria in L-form state provide. These include cell fusion and genome shuffling. It is significantly easier to fuse bacteria without cell walls interfering with the process. Fusion of L-forms is reasonably easy in the presence of PEG which will cause genetic recombination between fusants, forcing bacteria to reproduce sexually, and can be used for directed evolution.

We are also looking at the physical shaping of bacteria using microfluidic technology and insertion of L-form bacteria into plants. L-forms have been found growing naturally in plants and have been postulated to provide benefits in plants.

Themes

Shuffling Recombination and Endosymbiosis

Using L-form to perform genetic recombination has been shown to be 20X more efficient than Sexual PCR or any other current techniques. The advantages of using L-form are, it can grow, divide and re-integrate the cell form back. We are looking into two L-form fusion to potentially improve or generate novel functions; and try to create new bacteria strains via (cre-loxP system). Introducing foreign organisms (spores, smaller bacteria) into L-form and to see what happen to it, and triggering internal sporulation. We will also try to model the biophysical mechanism of the L-form cell membrane fusion.

Introducing and detecting L-forms in Plants

L-forms and plants can exist in a symbiotic relationship as plants provide an osmotically suitable environment. In return, L-forms can confer benefits to their host including reducing the rate of fungal infection. We plan to wash seedlings in a solution of GFP labelled L-forms, allowing the seedlings to take up the bacteria. We will then view our L-forms inside the plant using confocal microscopy.

In the future, L-forms could be engineered to supply nutrients to plants, potentially increasing crop yield in low fertility soil. L-forms are osmotically sensitive, giving the ethical advantage that they lyse if they escape from their host plant into the environment. Please click this box for more information.

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 and it's shape is governed by laws of physics, just like that of eukarytotic cells. The advantage of this is that 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 a hard-to reach target.

We will test this hypothesis by injecting the L-forms into specially designed microfluidics chambers and observe their behaviour under the microscope. Prior to the experiment we will attempt to construct a rough model of the membrane behaviour under stress conditions, which include cell growth and boundaries provided by the chamber walls.