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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. Initially we tested this idea using BioBrick BBa_J33204, encoding the reporter gene xylE encoding catechol-2,3-dioxygenase (which converts catechol, a cheap, colourless substrate, to bright yellow 2-hydroxy-cis,cis-muconic semialdehyde). We have previously demonstrated that this reporter gene works well in B. subtilis. 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). Chloramphenicol-resistant colonies were obtained, and the presence of xylE was confirmed by PCR, but no XylE activity was detected (ie, no visible yellow colour on addition of a drop of 10 mM catechol to the colonies). It was therefore concluded that BioBricks can be introduced into B. subtilis by this method, and that expression does not occur in the absence of a promoter.
The experiment was repeated using a composite BioBrick consisting of BBa_J33207 (lac promoter from E. coli) with BBa_J33204 (xylE reporter gene). Again, chloramphenicol-resistant colonies were obtained but no XylE expression was observed, suggesting that this promoter was not active in this context, even though the lacI repressor gene was not present.
To check that expression could be achieved, we finally turned to the only BioBricked native B. subtilis promoter in our freezer, BBa_J33206 (B. subtilis ars promoter, induced by sodium arsenate, cloned as part of the Edinburgh iGEM 2006 arsenic biosensor project. We made a composite BioBrick consisting of J33206+J33204, ligated this with pTG262 and transformed B. subtilis as above. 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).
A plasmid of the expected size was detected in plasmid DNA preps from this clone, but since the construct contains a significant amount of native B. subtilis sequence, we cannot at present exclude the possibility that the construct may have integrated into the genome at the ars locus. This can be tested by PCR to check the size of this locus.
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. Here are some sample data from an experiment in which the 'Bacillosensor' clone was incubated overnight in 50% v/v LB, 50% v/v water with 10 mg/l chloramphenicol and various concentrations of arsenic (as sodium arsenate, the most environmentally relevant form of this toxin). Incubation was at 37 C with shaking. The following morning, catechol was added to a final concentration of 0.5 mM and the samples were incubated at room temperature without shaking for several hours. Cells were then removed by centrifugation, and the absorbance at 377 nm (peak absorbance of the yellow product) of a 1/10 dilution was measured against a water blank.
- sterile growth medium: 0.027
- no arsenic: 0.048
- 10 ppb arsenic (WHO safety limit): 0.090
- 25 ppb arsenic: 0.117
- 50 ppb arsenic (Bangladesh safety limit): 0.184
- 75 ppb arsenic: 0.228
- 100 ppb arsenic: 0.309
The yellow colour was clearly visible by eye, making this potentially a useful addition to our range of arsenic biosensors, especially for use in developing countries, where the ability to store and distribute the organism in a dry form as spores will be especially useful.
