L-forms

BioBrick

Switch Brick

Purpose of BioBrick

Facilitate the switching of Bacillus subtilis cells from rod-shape to L-form, wall-less cells. Furthermore, this ‘switch’ should also enable L-form cells to return to rod-shape when required. Introduction to Project: There are two crucial changes that need to be introduced to the chromosome of B. subtilis to allow for functional L-form cells to develop:

1) Down-regulation in the production of peptidoglycan – a main component of the bacterial cell wall. The single point mutation leads to the overexpression of lipids that are essential for L-form growth.

2) Point mutation within the regulatory region of an ispA-like gene (yqiD) – a gene is involved in the formation of farnesyl pyrophosphate (FPP). FFP is an enzyme that is necessary for the formation of several essential lipids. This second change occurs via a spontaneous mutation that occurs in cells that survive the forced decrease in peptidoglycan production and loss of cell wall.

This switch will be facilitated through the introduction of a BioBrick that places a xylose-controlled promoter (PxylR) upstream of the murE gene in the B. subtilis chromosome. The murE gene is involved in the biosynthesis pathway of peptidoglycan.. Homologous recombination will allow this BioBrick to be integrated into the chromosome of B. subtilis.