IGEM:IMPERIAL/2008/Prototype/Chassis

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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B.subtilis Chassis

  • Chris D Hirst 13:37, 25 August 2008 (EDT):It seems the wiki is heading in a new organisational direction here, I've transfered the B.subtilis information currently present, if we discuss this tomorrow as a team and decide we want the chassis information separate (it's an increasingly major part of the project and personally I think it should be, rather than a large footnote), I'll flush this section out properly over the week

B.subtilis as a chassis made its first appearance last year and has many advantages for use at iGEM including (but not limited to!) a single membrane allowing for easier secretion, its high motility and a large amount of data in the literature. For our project, B.subtilis is vital. E.coli cannot secrete proteins to any high degree and modifications to it to allow this have not always proven particularly succesful. B.subtilis has however been used for this purpose for many decades and though there are some technical issues associated with this, it has performed the job admirably. B.subtilis is also highly motile and posses a unique (at present) system of switching off its motility in the form of a molecular clutch, EpsE (see Motility below).

To make B.subtilis a viable host, it is necessary to produce parts that will work correctly in B.subtilis. When searching the literature, it rapidly became clear that B.subtilis would not be able to use E.coli promoters with any real ability, would happily digest many E.coli produced vectors and also lacked a Ribosome capable of binding Ribosome Binding Sequences from E.coli. As such, we would need new vectors, new promoters and new RBSs in order to get the project off the ground.

To get round modifying a shuttle vector to meet Biobrick standards integration sequences can be used. These sequences are complementary to a gene in B.subtilis and may naturally be incorporated over those genes by the recombination system in B.subtilis. Promoters for B.subtilis were taken from both vectors and the organisms own chromosome via the extensive transcription regulation database for B.subtilis, the DBTBS, to suit the requirements of our system and to give a suitable range of basic promoters for B.subtilis. Although a consensus sequence (5'-AAGGAGGTG-3') for the B.subtilis RBS exists and has been proven to work effectively in the organism, two naturally occuring RBSs from B.subtilis that have been used extensively in the past were chosen as these are the most likey to work.


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