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Part:BBa_K1185000:Design

Designed by: Vincent Leonardo, Christopher Wall and Robert Mckiernan   Group: iGEM13_Newcastle   (2013-08-15)

B.subtilis Rod to L-form Switch


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2130
    Illegal BamHI site found at 308
    Illegal BamHI site found at 326
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

There were 3 restriction sites that did not meet the RFC10 requirements, two EcoRI sites and one PstI. The first EcoRI restriction site was located 218bp into the whole BioBrick (inside pbpb region), we changed the sequence from GAATTC to GCATTC. The second restriction site was located 2287bp into the BioBrick in the spacer region between PxylR and murE, we changed it from GAATTC to GTATTC. The PstI site was located 197bp into the BioBrick in the pbpb region and was change from CTGCAG to CTTCAG.


Source

All the sequences come from the Bacillus subtilis genomic sequence. The pbpb section in the BioBrick was taken from end of the pbpb gene 1841-2151bp (309bp). Sequence acquired through microbes online VIMSS38051. The murE section was taken from the start of the murE gene 1-331bp. Sequence from microbes online VIMSS38053. The cat gene and PxylR gene were both come from the B. subtilis genome, but can also be found in the BioBrick Registry (BBa_K823020 and BBa_K143014).


References

[http://www.ncbi.nlm.nih.gov/pubmed/22122227 Domínguez-Cuevas P, Mercier R, Leaver M, Kawai Y, Errington J. (2012) The rod to L-form transition of Bacillus subtilis is limited by a requirement for the protoplast to escape from the cell wall sacculus. Molecular Microbiology, 83, 52-66.]

[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.]

[http://www.ncbi.nlm.nih.gov/pubmed/11849491 Walker R., Ferguson CM., Booth NA and Allan EJ.(2002) The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea. Letters in Applied Microbiology. 34, 42-45.]