Contents 1 Switch BioBrick 1.1 Purpose and justification 1.2 Modelling 1.3 Construction 1.4 Cloning and integration 1.4.1 Competent cell preparation 1.4.2 Transformation 1.5 Testing and Characterisation 1.5.1 Liquid media 1.5.2 Lysozyme protoplasting method

Switch BioBrick

Purpose and justification

The purpose of this main ‘keystone’ biobrick is to facilitate the switching of Bacillus subtilis cells from rod-shape to L-form, wall-less cells. Furthermore, this ‘switch’ enables L-form cells to return to rod-shape when required.

This is facilitated through the introduction of a xylose-controlled promoter (PxylR) upstream of the murE gene, which is involved in the biosynthesis of peptidoglycan. The presence, or absence of a cell wall can be controlled through the presence, or absence of xylose, respectively.

The antibiotic resistance marker chloramphenicol acetyl-transferase (cat) is upstream of this promoter region to allow for the selection of cells, in which this system is integrated.

Homologous recombination allows this BioBrick to be integrated into the chromosome of B. subtilis. The cat and PxylR sequences are flanked by sequences that are homologous with regions of the B. subtilis chromosome, which will allows insertion at the intended loci. ~300bp at the 5’ end of the BioBrick is homologous with the end of the pbpB gene. Similarly ~300bp at the 3’ end of the biobrick is homologous with the start of the murE gene.

Modelling

Modelling biosynthesis of peptidoglycan, specifically involving manipulation of the murE gene. Currently undergoing completion…

Construction

The designed BioBrick was synthesised by DNA synthesis company DNA 2.0. The components within the BioBrick were as follows: -

Parts:

1. Biobrick prefix – RFC 10 standard.
2. 309bp of the end of the pbpB coding sequence.
3. Spacer.
4. Reverse and complement of chloramphenicol acetyl-transferase (cat) coding sequence (including native ribosome binding site (RBS) and promoter).
5. PxylR promoter.
6. Spacer.
7. RBS binding site for murE.
8. Spacer.
9. 331bp of the start of murE coding sequence.
10. Biobrick suffix – RFC 10 standard.

Produced using Gene Designer 2.0

Cloning and integration

1. The gene construct will be clone into pSB1C3, which is the only plasmid acceptable by iGEM for part submission.

2. The plasmid that is returned from DNA2.0 will be amplified in E.coli. The following protocol outlines the procedure by which this will take place:

Competent cell preparation

Transformation

Testing and Characterisation

Transformants will be selected through growth on LB+Chloramphenicol+0.5-0.8% xylose media. Only those which took up the DNA and integrate the DNA will survive.

To characterise this BioBrick, the B. subtilis is to be grown on a media without xylose and then the morphology of the cells will be checked under the microscope. Successfully transformed B. subtilis cells should have converted to L-form state (lost their cell walls). These are recognisable due to their rounded and non-uniform morphology. Cells may also vary greatly in size.

There are 2 main approaches to do this:

Liquid media

Lysozyme protoplasting method