Team:Newcastle

Our Project

Our project focuses on the creation and applications of L-forms: bacteria that grow without a cell wall. We propose L-forms as a novel chassis for synthetic biology. Our principle BioBrick switches Bacillus subtilis cells between rod-shape and L-form.

We will use microfluidics to attempt genome shuffling and shape-shifting. It is easier to fuse bacteria without cell walls. Fusion will cause genetic recombination, allowing directed evolution. We will put L-forms in moulds to observe if they adopt different shapes.

L-forms exist symbiotically in plants, which we will visualise by growing GFP labelled L-forms inside seedlings. L-forms could be engineered to supply nutrients to their host. L-forms are osmotically sensitive, giving biosecurity that they lyse if they escape from the plant.

As outreach we reflected upon our project's implications with stakeholders, created a BioGame for the public and developed a workshop for those new to modelling. Finally, we evaluated the relationship between synthetic biology and architecture.

Rod to L-form Switch BioBrick

An L-form is a bacterium that has no cell wall but is still able to multiply. Many species including Bacillus subtilis and Escherichia coli have an L-form. Losing the cell wall alters the characteristics of a bacterium creating a wealth of new applications for a bacterial species. In order to produce L-forms we have developed a BioBrick to allow the switching between the rod and L-form state of B. subtilis.

This BioBrick integrates into the B. subtilis chromosome by homologous recombination. It replaces the region containing the end of pbpb gene and the beginning of the murE gene (murE enables cell wall biosynthesis). However, it replaces the constitutive murE promoter with a xylose-inducible promoter (PxylR). When xylose is present cell wall biosynthesis is switched on, giving rod cells. When xylose is not present, the cells will become L-forms.

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Shape Shifting

The loss of the cell wall leaves L-forms protected by only a fluid cell membrane. This allows L-forms to adapt to shapes of cracks and cavities, and possibly squeeze through tiny channels to deliver cargo to otherwise inaccessible targets.

We will inject L-forms into specially designed microfluidics chambers and observe their behaviour and shape. We are modelling membrane behaviour under stress conditions, which includes cell growth and boundaries provided by the chamber walls.

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.

Genome shuffling

Genome shuffling is an innovative way to select for improvement in desired traits, including survival in harsh conditions and increased protein production. One method involves squashing two genetically similar cells together until they fuse. This results in the swapping of sections between each genome, producing two new genetic mosaics. These can then be assayed for desired traits, and the procedure repeated.

L-forms do not have cells walls so are comparatively easy to fuse using microfluidics. We have created BioBricks encoding green and red fluorescently labelled DNA binding proteins. Fusing one GFP and one RFP chromosome labelled L-form will allow us to visualize the swapping of DNA between the bacteria: the new cells will fluoresce with both red and green.