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.

Integration of the BioBrick facilitates xylose-mediated control over the expression of murE. This cascades through biosynthetic pathways to enable control over peptidoglycan synthesis and thus control over cell wall production. Simply, in B. subtilis cells that have integrated the BioBrick into their chromosome, only in the presence of xylose is the cell wall produced. When xylose is not present cells lose their cell wall and survivors adopt the L-form phenotype.

Design

Primers were designed to sequence the region between the pbpB and murE genes on the B. subtilis LR2 chromosome. These primers allowed sequencing from the 5’ end of the region containing the sequence required to facilitate switching between cell-walled form and L-form. The design of the primers allowed for sequencing to include the final 309bp at the 3’ end of the pbpB coding sequence - upstream of the L-form switch region.

 A second primer was designed to allow sequencing from the 3’ end of the L-form switch region.

This primer was complimentary to sequence within the murE gene and so sequencing included the reverse sequence of the 331bp at the 5’ end of the murE coding sequence; this gene is down-stream of, and in fact under the control of the L-form switch. The L-form switch sequence (including the final 309bp of the pbpB gene at the 5’ end of the sequence and the first 331bp of the murE coding sequence at the 3’ end of the switch sequence) was amplified from B. subtilis LR2 chromo-somal DNA via PCR using these designed primers. This amplified DNA was then sequenced by Geneius labs using the two designed primers. Fur-ther primers were designed to sequence internally within the L-form switch region, based upon the sequence data provided using the first two primers. Again sequencing was carried out by Geneius labs. The sequence for the L-form regulatory mechanism was obtained through a de novo assembly of the sequence data gained from sequencing the specific region between the pbpB and murE genes on the B. subtilis LR2 chromosome with reads produced by high throughput sequencing of the full B. subtilis LR2 genome. Specifically reads that did not assemble when using B. subtilis 168 as a reference genome were used in this assembly. Sequence assembly was carried out using Sequencher 5.1 DNA sequencing software. To allow the L-form switch synthetic construct to be submitted to the iGEM registry of standard biological parts – an open source catalogue of genetic ‘BioBricks’ – it needed to comply with BioBrick RFC (request For Comment)[10] standard. To be compatible with this standard a synthetic construct must not contain the restriction sites EcoRI, XbaI, SpeI, PstI or NotI. Additionally the synthetic construct must be appended with a specif-ic prefix, containing the EcoRI, NotI and XbaI sites, and a specific suffix, containing the SpeI, NotI and PstI sites. This prefix and suffix allow for the standard assembly of multiple BioBricks. To allow the L-form switch BioBrick sequence to be compatible with the BioBrick RFC[10] the prefix and suffix sequences were added to the synthetic construct sequence using Sequencher 5.1 DNA sequencing software. Two EcoRI restriction sites and a PstI restriction site that were present in the L-form switch sequence were removed through single nucleotide changes using Gene Designer 2.0. The bases used in these single nucleotide changes were chosen to optimise the altered codons (in the open reading frame of coding sequences) for codon bias in B. subtilis. Once the fully sequenced L-form switch synthetic construct was compatible with the BioBrick RFC[10] this sequence (see fig. 1.) was synthesised by DNA 2.0 into the pSB1C3 plasmid

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:

Biobrick prefix – RFC 10 standard.
309bp of the end of the pbpB coding sequence.
Spacer.
Reverse and complement of chloramphenicol acetyl-transferase (cat) coding sequence (including native ribosome binding site (RBS) and promoter).
PxylR promoter.
Spacer.
RBS binding site for murE.
Spacer.
331bp of the start of murE coding sequence.
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:

Team:Newcastle/Parts/l form switch

From 2013.igem.org

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