The PorA or class 1 protein is one of the major meningococcal outermembrane proteins (OMPs). It is one of the two porins found in this organism, the other one being the PorB or class 2/3 protein. It folds into a 16-stranded β-barrel structure, which is now well-established for bacterial porins, in which seven loops are exposed at the cell surface and the remaining one forms the constriction of the pore (1,2). There are approx 20 different serosubtypes of PorA (3), based on sequence variability in the longest surface-exposed loops 1 and 4 (see Fig. 1). In addition, minor sequence variations within individual subtypes have been observed. As a result, some subtypes such as P1.10 actually constitute a family of variants differing by single amino acid substitutions, which may affect antibody recognition; for other subtypes such as P1.4 the number of variants is more limited (4,5). Several studies with experimental outer membrane-derived vaccines have shown that PorA is a major inducer of bactericidal antibodies (6-8), making it a crucial component of any meningococcal vaccine. These antibodies are highly subtype-specific. Epidemic strains tend to be clonal and mainly express a single PorA subtype that changes only slowly over time (9). In hyperendemic situations, more variation is found but it is generally still possible to select a limited number of PorA subtypes that will cover most of the strains (10). However, PorA variation in both time and geography means that it is unlikely that a universal once-and-for-all meningococcal vaccine based on this protein alone can ever be made. This necessitates the use of vaccine strains with flexible PorA composition, in which new variants can be inserted into established production strains when required by new epidemiological circumstances. This chapter will describe methods to construct isogenic meningococcal strains with altered porA genes, which can be used both for vaccine production and as test strains to determine the precise epitope specificity of bactericidal antibodies directed against the various loops of PorA. Fig. 1. Topology model for PorA protein. Residues shown in boldface represent the surface-exposed P1.5 and P1.2 epitopes in loop 1 and 4. Residues marked with an asterisk represent the points of insertion into the KpnI site in loop 5 or 6.