Contents 1 L-forms 1.1 Overview 1.2 Purpose 1.3 Aims 1.4 References

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 [http://www.ncbi.nlm.nih.gov/pubmed/19212404 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).

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 BioKawaiBrick 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.

References

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603455/ Errington J. (2013) L-form bacteria, cell walls and the origins of life. Open Biology, 3, 120143.]

[http://www.ncbi.nlm.nih.gov/pubmed/19212404 Leaver M., Dominguez-CuevasP., Coxhead J.M., Daniel R.A. and Errington J. (2009) Life without a wall or division machine in Bacillus subtilis. Nature, 457, 849-853.]

[http://www.ncbi.nlm.nih.gov/pubmed/23452849 Mercier R., Kawai Y. and Errington J. (2013) Excess membrane synthesis drives a primitive mode of cell proliferation. Cell, 152, 997-1007.]