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Team:Edinburgh/Results/Bacillobricks
From 2008.igem.org
Bacillobricks: Introduction of BioBricks into Bacillus subtilis
Bacillus subtilis is potentially superior to E. coli as a host for some projects, for several reasons:
- It is much more effective at secreting proteins into the medium, as E. coli lacks the Main Terminal Branch of the General Secretory Pathway.
- As a Gram positive bacterium, it lacks the toxic lipopolysaccharide (endotoxin) of Gram negative bacteria such as 'E. coli
- The cells are considerably larger, making it easier to visualise intracellular components.
- B. subtilis forms endospores, a highly stable, heat and dessication resistant resting state which can be stored dry for years or decades, and will then germinate in less than 30 minutes when added to a suitable growth medium.
- B. subtilis is not pathogenic and has even been used as a probiotic organism in human foods.
However, standard BioBrick vectors do not allow introduction of BioBricks into B. subtilis. We felt that B. subtilis was potentially a suitable host for our 'Edinburgh Process' of conversion of cellulose to starch, due mainly to its ability to secrete enzymes such as cellulases. We therefore investigated two processes to allow introduction of BioBricks to B. subtilis, either on a plasmid or by integration into the genome. These experiments were carried out mainly by C. French (instructor) and Nimisha Joshi (advisor).
Introduction of BioBricks into B. subtilis on a plasmid
A standard method of transferring BioBricks between E. coli and B. subtilis would be the use of a shuttle vector which could replicate in both organisms, and in fact the Edinburgh iGEM 2007 team did demonstrate this using the Lactobacillus plasmid pTG262. pTG262 replicates in both E. coli and B. subtilis from the same versatile origin of replication, and has a multi-cloning site with EcoRI and PstI sites allowing convenient incorporation of BioBricks. pTG262 was submitted to the Registry as BBa_I742103, but our understanding, from one team that tried to acquire it from the Registry, was that it is not currently available as the transformation failed. Which brings us to the problem with pTG262 - it does not transform lab strains of E. coli with high efficiency, at least not in our hands. (For more information about pTG262 see http://www.openwetware.org/wiki/Cfrench:BioBrickVectors1).
To get around this problem, we decided to check whether we could ligate a BioBrick into pTG262 and then transform B. subtilis directly with the ligation mixture, rather than initially preparing DNA from E. coli. First we made a testable Bacillus BioBrick by combining two BioBricks from the Edinburgh 2006 arsenic biosensor project: BBa_J33026 (the arsenic-responsive promoter from B. subtilis and BBa_J33204 (the xylE reporter gene, which we have previously shown to work well in B. subtilis. The resulting BioBrick was prepared and checked in pSB1A2 with E. coli as a host, then the insert was cut out with EcoRI and PstI and ligated with pTG262 cut with the same two enzymes. The ligation was then used to transform B. subtilis using a standard procedure (see http://www.openwetware.org/wiki/Cfrench:BacTrans1) and cells were plated on LB with chloramphenicol (10 mg/l). Since the pSB1A2 vector band could not be separated from the Pars+xylE BioBrick on a gel (as they were the same size), it was expected that half of the colonies would contain the correct insert. In fact, one of four clones tested showed evidence of XylE activity (ie, a yellow pigment produced when a drop of 10 mM catechol was added to a colony) and presence of xylE by PCR (we have so far been unable to reliably prepare plasmid DNA from B. subtilis).
Interestingly, this clone showed highly sensitive arsenic-dependent induction of XylE activity and was capable of detecting arsenic at the WHO recommended threshold level of 10 ppb, unlike our previous attempts at non-BioBrick-based B. subtilis arsenic biosensors. This makes it potentially a useful biosensor for use in developing countries such as Bangladesh, where arsenic in groundwater is a major public health problem, since the biosensor can be stored and distributed in the form of dried endospores, which is not possible with E. coli-based biosensors.
