Orthogonality and burden of a heterologous AND gate gene circuit in E. coli host

RNA-Seq sample preparation and sequencing E. coli NCM3722 cultures were grown and growth stopped 4 hrs post day dilution by adding 1/10 volume of 5% phenol 95% ethanol (v/v). Cells were harvested by centrifugation (4500 x g for 30 min). Supernatants were discarded and pellets drained by gravity flow for 5 min. Pellets wet weights were measured by subtracting the weights of cognate empty tubes. There are 7 samples in total containing 6 different plasmid constructs in the E. coli host. Samples 1 and 2 are biological replicates of the same construct (AND gate in pSB3K3), Sample 3 (Inputs-gfp in pSB3K3), Sample 4 (empty pSB3K3), Sample 5 (AND gate in pSB4K5), Sample 6 (Inputs-gfp in pSB4K5), Sample 7 (empty pSB4K5). The pellet cell samples were frozen at -80 °C before sent out on dry ice to vertis Biotechnologie AG for RNA-Seq. In brief, the cell pellets were incubated with lysozyme for 15 min at room temperature. The total RNA was then isolated using the mirVana RNA isolation kit (Invitrogen) including DNase treatment. Primary transcript enrichment was achieved by rRNA depletion and treatment with Terminator exonuclease (Epicentre) to remove other processed RNAs. RNA was fragmented using RNaseIII and cDNA libraries were built including PCR amplification with barcoded sequencing adaptors. Samples were pooled in approximately equimolar amounts to form one cDNA pool. The cDNA pool was sequenced on an Illumina HiSeq 2000 machine. The short sequence alignment software "Bowtie" (19) was used to map RNA-Seq reads (about 20 million each sample) on the E. coli MG1655 annotated genome (NCBI accession number NC_000913) and the cognate plasmid circuit sequences of each sample. The number of mapped reads for each gene was determined according to their annotated location features (NCBI gff format). The expression levels of genes were subsequently determined using the normalized measure of RPKM (Reads Per Kilobase of transcript per Million mapped reads). Read mapping were visualized using the Integrative Genomics Viewer tool (IGV).