Detailed background information - B. subtilis

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[edit] Bacillus subtilis and genetic manipulation

  • B. subtilis is a Class I contaminant! Thus we don't need extra clearance to use it. The website [1] sourced this information from a paper, which is unfortunately not part of our subscription.
  • The paper detailing the sequencing of the genome of B. subtilis is described here [2]. More details here [3]
  • Codon usage differs from E. coli to some extent (see paper above - gives a nice overview), not sure whether that matters really much (I was thinking: systems like lux are not native to E. coli as well - and they work just fine)
  • This is absolutely priceless in terms of information on what we can do with bacillus. Unfortunately nothing on Agr circuits.
  • Bacillus subtilis is very often used in genetic engineering. It has been used in a multitude of experiments where it was used in the way one could expect E. Coli to be used. It also appears that Bacillus can be grown in the same way as E.Coli: 37C, in LB and induced with IPTG.
  • It seems that chromosomal insertion can be used in Bacillus. [4]
  • This is quite interesting [5]: they characterised a promoter that has been tested in Bacillus subtilis that can be inserted into the bacterial chromosome and then start expression of heterologous proteins.
  • This demonstrates tagging of Bacillus gene expression by fluorescence.
  • Requirements for Transformation in Bacillus subtilis, Anagnostopoulos and Spizizen et. al., J Bacteriol 1961. [6]
    • The granddaddy of all transformation protocols, used in many B. subtilis labs worldwide since time of publication!

[edit] Possible promoters that can be used in B. subtilis and E. coli

[edit] Promoters which aren't BioBricked

  • Function of Corynebacterium glutamicum promoters in Escherichia coli, Streptomyces lividans, and Bacillus subtilis, Patek et. al., J Biotech 2003. [7]
    • Mentions that the promoters P-per, P-45 and P-aes 1 + P-aes 2 can achieve significant activities in B. subtilis and E. coli
    • Quoted from paper: "However, there are also differences among promoters recognized by major sigma factors responsible for the transcription of the prevailing category of genes, called house-keeping genes. Accumulated evidence indicates that these differences are based particularly on the evolutionary distance between the species and generally on the different G/C content in the respective genomes. Gene expression in bacterial genera with high A/T content (Streptococcus , Lactococcus, Bacillus) requires mostly high similarity of the core promoter sequences (-10 and -35 regions) with the consensus sequence. In contrast, promoters in bacteria with high G/C content (Streptomyces, Mycobacterium) tolerate a much higher variability within the consensus sequence elements." (I am inclined to believe that E. coli, with a G/C content of 52%, belongs to the latter category)
  • Efficient Utilization of E. coli transcriptional signals in B. subtilis, Peschke et. al., J Mol Biol 1985. [8]
    • Transcriptional machinery in vegetative state of B. subtilis closely resembles that of E. coli
    • Thus, not too surprising that B. subtilis promoters are efficiently utilized by E. coli RNA polymerase (corroborated by Expression in Escherichia coli of Bacillus subtilis tRNA Genes from a Promoter within the tRNA Gene Region, Vold et. al., J Bacteriol 1986. [9])
    • However, the reverse situation (E. coli promoters in B. subtilis), in general, does not seem to work
    • This paper finds that for E. coli promoters that are efficiently expressed in B. subtilis, the sequence immediately upstream of the -35 region is A/T rich
      • Promoters that support this finding are: veg, vegII (highest activity) and T5/T7 bacteriophage promoters
    • Other promoters that work well, but doesn't seem to have high A/T content are tac, trp, and lacUV5
    • There are some promoters with high A/T content but aren't utilized well; the authors of the paper writes that there is a "subtle interplay between RNA polymerase and multiple regions within the promoter".
    • Spacer region between the -35 and -10 region is almost always 17nt in E. coli; however B. subtilis seems to tolerate 16-18nt gaps well
    • Mentions that at the time of writing, RBSes and terminators of E. coli are yet to be well characterized when used in B. subtilis
      • However, they did try to use E. coli terminators in B. subtilis, and it sort of worked (different activity levels)
      • Let's hope that B0015 works across species!
  • Effect of -16 deletions on transcriptional efficiency of amyP promoter
    • in vitro transcription from amyP promoter using E.coli and B.subtilis polymerase and plasmid is pAMY10.
    • Note!: amyP2 promoter found to work in vitro in B.subtilis but not in vivo!
    • features of B.subtilis promoters: A- and T-rich regions upstream of the –35 region and A residues just downstream of the –10 region as well as a -16 region (TRTG)
    • TRTG compensates for the lack of a –35 region (33), while in promoters with a –35 region the –16 region may function in an additive manner to promote or stabilize a rate limiting initiation step
    • It is thus vital for transcription by E.coli RNAP from ‘extended –10 promoters’ and from the B.subtilis a-amylase promoters when placed in E.coli
  • Article on B.subtilis sigma factors
    • sigmaA is the B. subtilis counterpart of sigma70 and, as such, likely to direct the transcription of most of the B. subtilis genes expressed during growth in rich medium. In addition to expressing these "housekeeping genes", sigmaA is also involved in specialized gene expression
  • The -16 region of Bacillus subtilis and other gram-positive bacterial promoters, Voskuil et. al., Nucleic Acids Research 1998. [10]
    • This paper demonstrates that the amyP/amyP2 promoter works in both E. coli and B. subtilis. The wild-type plasmid is pAMY10.
    • Actual sequence of the amyP/amyP2 promoter here

[edit] Promoters which are BioBricked

  • Integrative elements for Bacillus subtilis yielding tetracycline-dependent growth phenotypes, Bertram et. al., Nucleic Acids Research 2005. [11]
    • Uses transposons to shove the Tet operator into B. subtilis, and some strains showed tetracycline-dependent growth
    • They used tetracycline operators (ie, the TetR promoter, I think)
    • Maybe we can introduce the constitutive mRFP (BBa_I13521) into B. subtilis and check for red colonies!
  • Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis, Yansura et. al., Proc. Natl. Acad. Sci. USA 1984. [12]
    • Placed the lac operator (site where lacI repressor binds) after a B. subtilis promoter to create a hybrid promoter responsive to IPTG/allolactose, expression can thus be induced
    • Completely novel way to control expression in B. subtilis (usually gene regulation in B. subtilis achieved by varying sigma factors in B. subtilis)
    • Did not explain why they preferred to create a hybrid promoter than just using the lac promoter
    • Sequence of lac operator is AATTGTGAGC GGATAACAAT TCCG
    • Still worth a try to introduce the constitutive GFP promoter (BBa_J04430) to test whether the lac promoter works in B. subtilis

[edit] Software for predicting promoter sites

(see also Useful software)

[edit] Shuttle vectors

  • A series of shuttle vectors for Bacillus subtilis and Escherichia coli, Bruckner et. al., Gene 1992. [13]
    • Describes the construction of pRB373, pRB374 shuttle vectors with the B. subtilis pUB110 origin and the E. coli pBR322
    • With these genes, cloning and expression of genes as well as probing of regulatory signals can be performed in B.subtilis and E.coli
  • Protein expression from an Escherichia coli/Bacillus subtilis multifunctional shuttle plasmid with synthetic promoter sequences, Trumble et. al., Protein Expression and Purification 1992. [14]
    • Describes pSP10, a shuttle vector with the following characteristics:
      • A selection gene - chloramphenicol acetyltransferase
      • An indicator gene encoding β-galactosidase for visual identification of colonies containing DNA inserts
      • A cloning region immediately upstream from the indicator gene
      • Origins of replication recognized by both Escherichia coli and Bacillus subtilis
      • A synthetic DNA expression control sequence, including -35 and -10 regions, ribosomal binding site, and transcriptional and translational start sites.

[edit] Mutants of interest

  • Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases, Wu et. al., J Bacteriol 1991. [15]
    • Created a strain of B. subtilis WB600, with mutations to 6 of the extracellular proteases, which showed only 0.32% of the wild-type extracellular protease activity
    • There are already improvements to this initial strain, imaginatively called WB700 (7 mutations) and WB800(8 mutations)
  • Proteomics of Protein Secretion by Bacillus subtilis: Separating the "Secrets" of the Secretome, Tjalsma et. al., Microbiology and Molecular Biology Reviews 2004. [16]
    • Used WB700 in this paper, and they claim that "consistent with the idea that homologous secretory proteins must be largely resistant to the extracellular proteases of B. subtilis, the levels of most extracellular proteins were not affected by the seven protease mutations."
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