IGEM:IMPERIAL/2008/Projects/B. subtilis Chassis

Chassis

To B.subtilis or not to B.subtilis, that is THE question

The publishing of the B.subtilis genome[1] may allow simple ways to obtain sequence for potentially useful promoters for the B.subtilis chassis

Vector

E.coli plasmids cannot replicate in B.subtilis and so regular biobricks are not usable in such a chassis.

In labs, constructs called shuttle primers capable of replicating in both species are used and often created by merging together an E.coli vector and a B.subtilis vector. One such B.subtilis vector is pC194[2].

pC194 requires a DNA sequence approxiamtely 1300bp[3] in length that could be potentially cloned into the regular biobrick vector

Given the activity of B.subtilis degrading E.coli derived plasmids however. It may be possible to utilise PCR for plasmid production and selection of transformants in B.subtilis to by-pass this issue, or to produce the plasmid in B.subtilis which would be more difficult.

Thonly other option would be to use the Cambridge 2007 approach and attempt to produce a modified shuttle vector to BioBrick standard

Promoters

Constitutive promoters:

Use of basal transcription promoters, potentially the promoter for one or various rRNAs, P3 promoter, RNAP subunit gene promoters and metablic gene promoters.

P3 promoter

Annotated B.subtilis genome

Genome project

Metabolic Pathways of B.subtilis

Probably ideal to pick a few (say 5) and characterise in order to find relative levels for use

1 rRNA promoter

1+ Metabolic related promoter (potential inducibility)

1 RNAP subunit promoter

P3 promoter

Another basaly transcribe sequence

All non-constiutive promoters should remain functional in B.subtilis though leaky (basal) transcription rate will however be different, the key promoter is the one at the start of the chain...

RBSs?

Needs research...

Reference

1. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V, Bertero MG, Bessières P, Bolotin A, Borchert S, Borriss R, Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S, Bruschi CV, Caldwell B, Capuano V, Carter NM, Choi SK, Cordani JJ, Connerton IF, Cummings NJ, Daniel RA, Denziot F, Devine KM, Düsterhöft A, Ehrlich SD, Emmerson PT, Entian KD, Errington J, Fabret C, Ferrari E, Foulger D, Fritz C, Fujita M, Fujita Y, Fuma S, Galizzi A, Galleron N, Ghim SY, Glaser P, Goffeau A, Golightly EJ, Grandi G, Guiseppi G, Guy BJ, Haga K, Haiech J, Harwood CR, Hènaut A, Hilbert H, Holsappel S, Hosono S, Hullo MF, Itaya M, Jones L, Joris B, Karamata D, Kasahara Y, Klaerr-Blanchard M, Klein C, Kobayashi Y, Koetter P, Koningstein G, Krogh S, Kumano M, Kurita K, Lapidus A, Lardinois S, Lauber J, Lazarevic V, Lee SM, Levine A, Liu H, Masuda S, Mauël C, Médigue C, Medina N, Mellado RP, Mizuno M, Moestl D, Nakai S, Noback M, Noone D, O'Reilly M, Ogawa K, Ogiwara A, Oudega B, Park SH, Parro V, Pohl TM, Portelle D, Porwollik S, Prescott AM, Presecan E, Pujic P, Purnelle B, Rapoport G, Rey M, Reynolds S, Rieger M, Rivolta C, Rocha E, Roche B, Rose M, Sadaie Y, Sato T, Scanlan E, Schleich S, Schroeter R, Scoffone F, Sekiguchi J, Sekowska A, Seror SJ, Serror P, Shin BS, Soldo B, Sorokin A, Tacconi E, Takagi T, Takahashi H, Takemaru K, Takeuchi M, Tamakoshi A, Tanaka T, Terpstra P, Togoni A, Tosato V, Uchiyama S, Vandebol M, Vannier F, Vassarotti A, Viari A, Wambutt R, Wedler H, Weitzenegger T, Winters P, Wipat A, Yamamoto H, Yamane K, Yasumoto K, Yata K, Yoshida K, Yoshikawa HF, Zumstein E, Yoshikawa H, and Danchin A. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997 Nov 20;390(6657):249-56. DOI:10.1038/36786 |
2. Baudet MF, Dachet C, and Beaumont JL. In vitro interaction of LDL, anti-lipoprotein IgA and human fibroblasts. Biomedicine. 1978 Oct;29(6):217-20.

3. pmid=

4. Kolling GH and Schratz B. [Comparative studies of twilight vision with the Mesoptometer I and II and the Nyktometer]. Fortschr Ophthalmol. 1991;88(2):178-81.