Overview of the Brucella melitensis ORFeome construction. The main experimental steps of the ORFeome construction, that is, PCR, BP cloning, transformation, and validation of the entry clones, are shown in the top part of the figure. The success/failure distribution is given for the PCR, the presence of transformants after BP reaction, and the validation of the entry clones. The success rates are represented with hatched surfaces, whereas failure rates are shown in black. For the last step, the additional portion (vertically hatched surface) corresponds to entry clones checked by PCR using ORF-specific primers (see text for details). B1 and B2 stand for attB1 and attB2, respectively, and L1 and L2 correspond to attL1 and attL2 sites, respectively.