Figure 4

Enrichment protocol for the selection of mutations in InlA conferring enhanced invasion of L. lactis into CT-26 cells. Cultures of L. lactis + pNZB containing (i) inlA^WT (ii) inlA^m* or (iii-vi) 4 banks of clones with different levels of mutation in the LRR of inlA^WT were induced with nisin and assayed for invasion into CT-26 cells by gentamicin protection assay. The residual lysate from invaded CT-26 cells inoculated into GM17 containing Cm 5 μg/ml and grown overnight, the culture was then frozen at -80°C. The entire process was repeated with the frozen stock serving as the seed for the inoculum.