Team:Newcastle/Project

Overall project

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.

We are working on four themes which include: Shuffling, Recombination & Endosymbiosis; Introducing and detecting L-forms in Plants; Shape-shifting; Investigating two-component systems in L-forms.

Project Details

Shuffling, Recombination & 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

It is thought that L-forms exist in plants in a symbiotic relationship. The plants provide conditions for L-form growth and L-forms may offer advantages to the plant, such as protection from pathogens. In the future it may be possible to genetically engineer L-forms so that, on introduction into a plant, they provide their host with beneficial substances. We aim to illustrate that L-forms can survive in plants by two methods:

1) We will insert the gus reporter gene into L-forms. Grow L-forms in plants and visualize L-forms by the addition of X-gluc.

2) We will produce L-forms which express Red Fluorescent Protein and grow them in plants.

Shape-shifting

Our aim is to shape the bacteria to fit different molds using the microfluidics system, model the biophysical characteristics of bacterial cell membrane, and see what happens when we switch the cell wall synthesis back on.

Investigating two-component systems in L-forms

Two component systems form a pathway of communication found within bacteria. These rely upon two proteins - a sensor kinase, and a response regulator. The sensor kinase is embedded in the membrane and binds signalling molecules that are external to the cell. Once triggered by external signals, the sensor kinase interacts with the response regulator - an intracellular protein. The response regulator is then able to migrate to gene regulatory regions of DNA - promoters - to influence transcription of specific genes.