Contents 1 L-forms 1.1 Overview 1.2 Purpose 1.3 Aims

L-forms

Overview

We propose the use of L-form bacteria as a chassis for synthetic biology. L-form strains are derivatives of common cell-walled bacterial strains; however, L-forms are cell wall deficient. Many modern bacteria have the capacity to switch into L-form state, though specifically we are investigating L-forms in the model Gram-positive bacteria Bacillus subtilis. Unlike protoplasts, L-forms are still able to propogate and grow like their cell-walled counterparts. This growth and division does not occur in the same way as walled cells - L-forms have been described to undertake [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603455/ membrane blebbing, tubulation and vesiculation].

As a result of their cell wall-deficiency L-forms are restricted by osmolarity much more then walled bacteria. L-forms require incubation for many generations in specific osmotically balanced conditions with enzymes and antibiotics that prevent invasion by walled contaminant cells. However, these specific osmotic demands of L-forms can also be seen as a kill-switch. Any bacteria that escape will no longer be in a maintained and osmotically balanced environment and will consequently not survive.

For conventional cell-walled bacterial strains to switch into wall-deficient L-form state the synthesis of the peptidoglycan cell wall must be disrupted. In B. subtilis this disruption can be achieved through controlling the expression of murE. The murE gene is responsible for the synthesis of a number of enzymes that are involved in the synthesis of a peptidoglycan precursor. When murE expression is down-regulated, this has a cascade effect leading to the down-regulation of peptidoglycan synthesis. It has been found that a mutation that spontaneously occurs in L-form B. subtilis cells is also necessary for the survival of stable L-forms (Leaver et al.2009). After selection for wall-deficient cells, survivors exhibit a mutation within the yqiD gene, which is similar to the Escherichia coli gene ispA. This mutation allows [http://www.ncbi.nlm.nih.gov/pubmed/23452849 for stabilisation of L-forms that are undergoing shape modulation due to excess of cell membrane] (Mercier et al. 2013).

A potential chassis for use in the future of synthetic biology is that of L-form bacteria. L-form strains are derivatives of common bacterial strains that do not possess a cell wall (Leaver et al. 2009). Interestingly, many modern bacteria are capable of switching into wall-deficient L-form state (Allan et al. 2009). They differ from protoplast bacteria in that L-forms are able to propagate, grow and divide – once the cell wall is lost in regular protoplasts this does not occur. Growth and division by L-forms occurs via a number of processes that involve ‘membrane blebbing, tubulation, vesiculation and fission’ (Errington 2013). Due to their specific osmotic demands, growth of L-forms requires incubation for many generations on precise osmotically protective media with antibiotics and enzymes that eliminate walled contaminant cells (Leaver et al. 2013). Importantly the characteristics of L-forms suggest the potential for their use as a chassis for synthetic biology. Protoplast cells transform more easily than walled bacteria (Chang and Cohen 1979) with the obstacle of the cell wall removed, the same is true of L-forms. Furthermore, the specific osmotic requirements of L-form bacteria acts as a kill-switch, greatly lowering the risk of escape of genetically-modified organisms – an ethical concern raised in relation to synthetic biology. The loss of the cell wall in L-forms also makes them useful for horizontal gene transfer via cell fusion, as has been carried out in protoplasts (Hopwood et al. 1977) and plants (Kao and Michayluk 1974). This can be useful for genome shuffling and directed evolution. In order for a conventional cell-walled strain to switch into wall-deficient L-form state, synthesis of the peptidoglycan cell wall must be interrupted and synthesis of excess cell membrane achieved. In B. subtilis LR2 (Mercier et al. 2013) this switch is achieved through two genetic changes to the chromosome of a B. subtilis 168 derivative. To interrupt the synthesis of peptidoglycan a xylose-dependent Pxyl promoter was introduced into the chromosome to control murE expression. The murE gene encodes numerous enzymes necessary for peptidoglycan precursor synthesis (Dominguez-Cuevas et al. 2012). Cells containing this Pxyl promoter could be selected for using chloramphenicol – a cat (chloramphenicol resistance gene) was inserted with the Pxyl promoter, upstream of murE. The cat gene and Pxyl were inserted in place of the spoVD gene – involved in sporulation, and were thus located between the pbpB and murE genes on the chromosome. A mutation that spontaneously occurs in L-form B. subtilis LR2 cells that survive selection (against walled cells) within the yqiD gene (similar to the Escherichia coli gene ispA) is also necessary for stable L-forms (Leaver et al.2009). This mutation functions through stabilising L-forms that are undergoing shape modulation due to excess cell membrane (Mercier et al. 2013).

Purpose

To create a BioBrick that enables the conversion of cell-walled B. subtilis cells into L-form cells that are cell wall deficient. The BioBrick should also facilitate the reversion back to walled cells.

Aims

• Design a BioBrick which places murE under the control of a controllable promoter.

• Have this designed BioBrick synthesised.

• Integrate the designed BioBrick into B. subtilis.

• Determine the functionality of the BioBrick in removing the cell wall.