Collateral fitness effects of mutations

We performed RNA-seq experiments to compare the gene expression profiles of cells expressing TEM-1 beta-lactamase with single-codon substitutions in the absence of beta-lactam antibiotics. Mutations with deleterious fitness effects in the absense of antibiotics also caused significant changes in gene expression, primarily in the induction of specific outer envelope stress response pathways and, in some cases, the mild-induction of a few genes in the heat-shock response pathway. Overall design: E. coli cells harboring single-codon substitutions to the TEM-1 gene were grown in monoculture growth assays. Controls consisted of cells with the wildtype TEM-1 allele that were not induced with IPTG as well as cells containing a ?TEM-1 gene deletion that were induced with IPTG. Growth was halted at about 330 minutes to ensure the cells were still in exponential growth phase based on OD measurements. Total RNA was extracted from pelleted cell cultures. mRNA was enriched and used to synthesize double-stranded cDNA. The double-stranded cDNA was end prepped and purified. Illumina adaptor sequences were ligated to the double stranded cDNA and PCR enriched before being purified and pooled for Illumina Tru-Seq (2 x 75 bp reads).