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

B.Subtilis related databases

• DBTBS: a database of transcriptional regulation in Bacillus subtilis
• Non-Redundant B.subtilis database
• Swiss-Prot B.Subtilis
• SubtiList (Pasteur France)
• BSORF: Bacillus subtilis Genome Database (Japan)
• Micado

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

Assay and characterisation of a strong promoter element from B. subtilis [4]. Despite the pigdin English this paper describes the identification of a strong promoter element from B. subtilis - the authors already put it into an expression vector. It is stronger than the P43 promoter and is perhaps one we could make use of for constitutive expression?

A derepression system based on the Bacillus subtilis sporulation pathway is described [5] - an example of an engineering approach to a derepression-based control of gene expression in B. subtilis and an example of a heterologous secreted protein being expressed into the medium and subsequent assaying for that secreted protein.

Use of basal transcription promoters, potentially the promoter for one or various rRNAs, P3 promoter[6], RNAP subunit gene promoters and metablic gene promoters.

Annotated B.subtilis genome

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...

RBS

On a preliminary basis, there appears to be some issues realted to the B.subtilis RBS, particularly the sequence and also codon usage with the normal AUG start becoming UUG in at least some B.subtilis vectors[7]

The codon usage in B.subtilis indicates that UUG encodes leucine not methionine and as such the UUG methionine found on the B.subtilis vector[8], indicating the issue may be confined to just the start codon

This will need looking into if B.subtilis is to be used

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. Horinouchi S and Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815-25. DOI:10.1128/jb.150.2.815-825.1982 |
3. Dagert M, Jones I, Goze A, Romac S, Niaudet B, and Ehrlich SD. Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage. EMBO J. 1984 Jan;3(1):81-6. DOI:10.1002/j.1460-2075.1984.tb01764.x |
4. Zhang AL, Liu H, Yang MM, Gong YS, and Chen H. Assay and characterization of a strong promoter element from B. subtilis. Biochem Biophys Res Commun. 2007 Mar 2;354(1):90-5. DOI:10.1016/j.bbrc.2006.12.137 |
5. Nijland R, Veening JW, and Kuipers OP. A derepression system based on the Bacillus subtilis sporulation pathway offers dynamic control of heterologous gene expression. Appl Environ Microbiol. 2007 Apr;73(7):2390-3. DOI:10.1128/AEM.02266-06 |
6. Leelakriangsak M and Zuber P. Transcription from the P3 promoter of the Bacillus subtilis spx gene is induced in response to disulfide stress. J Bacteriol. 2007 Mar;189(5):1727-35. DOI:10.1128/JB.01519-06 |
7. McLaughlin JR, Murray CL, and Rabinowitz JC. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283-91.
8. Shields DC and Sharp PM. Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. Nucleic Acids Res. 1987 Oct 12;15(19):8023-40. DOI:10.1093/nar/15.19.8023 |