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| =Chassis=
| | Moved: See the [[/Subtilis_Chassis|Subtilis Chassis]] page. |
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| To ''B.subtilis'' or not to ''B.subtilis'', that is THE question
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| The publishing of the ''B.subtilis'' genome<cite>#1</cite> may allow simple ways to obtain sequence for potentially useful promoters for the ''B.subtilis'' chassis
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| ==Vector==
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| ''E.coli'' plasmids cannot replicate in ''B.subtilis'' and so regular biobricks are not usable in such a chassis.
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| 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<cite>#2</cite>.
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| pC194 requires a DNA sequence approxiamtely 1300bp<cite>#3</cite> in length that could be potentially cloned into the regular biobrick vector
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| 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.
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| Thonly other option would be to use the Cambridge 2007 approach and attempt to produce a modified shuttle vector to BioBrick standard
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| ==Promoters==
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| Constitutive promoters:
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| Use of basal transcription promoters, potentially the promoter for one or various rRNAs, P3 promoter<cite>#4</cite>, RNAP subunit gene promoters and metablic gene promoters.
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| [http://www.ncbi.nlm.nih.gov/sites/entrez?dispmax=1&db=nucleotide&doptcmdl=graph&term=50812173&from=9300&to=20924| Annotated ''B.subtilis'' genome] | |
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| [http://www.genome.jp/kegg/atlas/metabolism/2/| Metabolic Pathways of ''B.subtilis'']
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| Probably ideal to pick a few (say 5) and characterise in order to find relative levels for use
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| ::1 rRNA promoter
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| ::1+ Metabolic related promoter (potential inducibility)
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| ::1 RNAP subunit promoter
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| ::P3 promoter
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| ::Another basaly transcribe sequence
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| 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...
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| ==RBS==
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| 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 becoming UUG in ''B.subtilis''<cite>#5</cite>
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| This will need looking into if ''B.subtilis'' is to be used
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| ==Reference==
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| <biblio>
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| #1 pmid=9384377
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| #2 pmid=6950931
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| #3 pmid=6323171
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| #4 pmid=17158663
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| #5 pmid=6793593
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| </biblio>
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