Quantitative Real-Time PCR Analysis

Total RNA for quantitative reverse transcriptase PCR (qPCR) was extracted as described for next-generation sequencing and analysis. Two-step quantitative real-time PCR (qPCR) was conducted using LightCycler ® FastStart DNA Master PLUS SYBR Green I (Roche Life Science) on a Light Cycler 480 Real-Time PCR 3System (Roche Co., Germany) and was performed by Welgene Biotech Co., Ltd. (Taipei, Taiwan). The four genetic regions involved in n-butanol production pathway (thil, crt-bcd-etfB-etfA-hbd, adhe) and ROS-targeted scavenging (ompC-tmt) were analyzed. R16s (rrsA gene) was used as a reference in all analyses. The primers are provided in Additional file 3: Table S2 in the supplemental material. For the relative quantification of gene expression, the comparative CT method was employed. The averaged CT was subtracted from the corresponding averaged r16S value for each sample; resulting in ∆CT. ∆∆CT was obtained by subtracting the average control ∆CT value from the average experimental ∆CT. The fold increase was determined by calculating log2 (2^−∆∆CT) for the experimental vs. control samples.