Metris et al. BMC Genomics 2011, 12:535 http://www.biomedcentral.com/1471-2164/12/535 on different carbon source was measured [32], and, other literature data [37,42,43,72]. Due to the scarcity of data in the literature, the model could only be tested with oxygen for respiration. Prediction of essential genes by FBA FBA consists of the computation of the possible fluxes, ν, going through the reactions of the metabolic network at steady-state. The system of equations is defined by the stoichiometric matrix, S, containing the stoichiometric coefficients of the metabolic reactions, with m being the number of metabolites, and n the number of fluxes [15]. At steady-state (i.e. during balanced growth when the biomass composition is assumed to be constant), S.ν = 0. There are more fluxes than metabolites (n >m), so the system is underdetermined. The fluxes are bound by thermodynamic feasibility so the space of solutions is a convex space [15]. It has been shown with some organ- isms, notably with E. coli [16], that the flux through the biomass is optimised for the uptake of nutrients during balanced growth, so the biomass equation can be used as an objective function to reduce the space of solutions. With less well characterised micro-organisms like C. jejuni, the biomass composition is not known quantita- tively. The biomass composition was assumed to be the same as for H. pylori except for (a) vitamin B6, which was added to the equation as the genome is predicted to encode the entire biosynthetic pathway bar one gene and (b) thiamine, for which the active form was assumed to be thiamine diphosphate rather than thiamine. Exploration of the space of solution: Even after appointing an objective function, there may be more than one solution to the optimisation problem. These solutions are referred to as silent phenotypes as the growth rate is the same but the internal organisation of the fluxes is different [73]. Due to the design of the algorithm, the solution returned by simplex linear pro- gramming generally minimizes the number of fluxes. To estimate whether a gene is essential or not, the ratio, Gr, of the biomass flux when the gene is absent to the biomass flux when the gene is present was calcu- lated in a given environment [74]. A gene was consid- ered essential if Gr <= 10-9 (arbitrary value). Medium composition: Minimal medium was used to validate the model. Its substrates were derived from the BIOLOG medium experiments http://www.biolog.com. The experiments to screen for essential genes were car- ried out in Brucella medium which is a rich medium con- taining pancreatic digest of casein, peptic digest of animal tissues, dextrose, yeast extract, sodium chloride and sodium bisulfite [75]. Since the composition of this med- ium is unknown, the medium was assumed to be similar to yeast extract [76], the composition used for simula- tions is indicated in the table in the Additional file 5: Page 12 of 14 medium composition. The chemicals are allowed to enter or leave the system through exchange reactions. The exchange fluxes of the carbon source were fixed to a maximum of 20 mmol/g dry weight of biomass/h, which is close to a maximum uptake rate for E. coli [77] and 5 mmol/g dry weight of biomass/h for oxygen, which is about a fourth of the maximum fluxes measured in air for E. coli as C. jejuni is microearophilic. The other nutri- ents present in the medium were assumed to be non-lim- iting with an arbitrary uptake higher boundary of 1,000 mmol/g dry weight of biomass/h. All the calculations related to FBA were carried out with the COBRA Toolbox [74] in Matlab (version R2010b, Mathworks, Inc.) equipped with the glpk solver for linear programming [78]. Transposon mutagenesis Two in vitro transposition libraries were constructed using mariner transposase, essentially as described by Gaskin and van Vliet [79] and Tn7 transposase from New England Biolabs with C. jejuni NCTC11168 geno- mic DNA. These were introduced into C. jejuni NCTC 11168 cells by natural transformation [79] and plated onto Blood Agar Base no.2 (Oxoid) plates supplemented with 5% v/v defribinated horse blood and kanamycin 50 μg/ml. After ~48 hours incubation at 42°C under micro- aerophilic conditions (5% oxygen, 10% carbon dioxide, 85% nitrogen) colonies were pooled and genomic DNA extracted using QIAgen genomic-tips (QIAgen). Mapping of transposon insertions Genomic DNA from the pooled colonies was used as template in polymerase chain reactions using a transpo- son specific primer and individual gene specific primers. Briefly, 50 μl reactions were set up using 100 ng geno- mic DNA, 10-50 pmol of each primer and 25 μl of Hot- StarTaq mix (QIAgen). Cycling conditions were 95°C for 15 min, followed by 30 cycles of 95°C 30 sec, 50°C 30 sec, 72°C 90 sec and a final 72°C 15 min extension step. Aliquots from each reaction were run on 0.8% agarose gels, which were stained with ethidium bromide. Gel images were captured using a GeneDoc system (Anachem). The sizes of observed bands were calculated using Labimage (Kapelan Bio-Imaging GmbH) and this data was inputted into Excel (Microsoft). Based on the transposon study, 47 insertional inactivation mutants were created using conventional methods to validate the Tn7/Mariner findings. Additional material Additional file 1: Tables with the reactions included in the model, the metabolites and the references use to reconstruct the model. In the reaction table, the EC numbers of the reactions are indicated as well