## 2 Materials and Methods
### RNA Isolation, cDNA Synthesis and Gene Expression  
To monitor the expression of metabolite and regulatory genes involved in PHZ and PHA production, quantitative real-time PCR (qPCR) was employed. The expression of _phaC1,phaZ,phaC2,phaD,phaF_ and _phal_ (all associated with PHA production) was determined in _P. chlororaphis_ PA23 and its derivative strains. The housekeeping gene, _rpoB_, was chosen to normalize expression values of the target genes. Expression of these genes in RMM glucose medium (supporting PHZ production) or RMM octanoic acid medium (supporting PHA production) was also compared. Relative gene expression in comparison to Wt P. _chlororaphis_ PA23 was calculated. Primers used for gene expression analysis are listed in Table 1. Primers were designed based on the sequences of the respective genes obtained from _P. chlororaphis_ PA23 (GenBank search accession no. NZ_CP008696).  
Cultures were grown in RMM (2% glucose) or RMM octanoic (20 mmol/L) at 30 \({}^{\circ}\)C for 24 h or 20 h, respectively. A sample (0.2 mL) for each time point was mixed with 0.2 mL RNA protect, vortexed, and then stored at \(-80\)\({}^{\circ}\)C until required for RNA isolation. Total RNA was extracted from the cell pellets using a PureLink RNA Mini Kit (Ambion, Life Technologies, Carlsbad, CA, USA) following the supplier's protocol. Residual DNA was removed through on column DNase treatment using the Pure link DNase system (Thermo Fisher Scientific, Waltham, MA, USA). The concentration of RNA was determined using a Nanodrop 1000 spectrophotometer (Thermo fisher Scientific, Waltham, MA, USA), and RNA integrity was analyzed by electrophoresis using an Experion system (Bio-Rad Laboratories Canada, Mississauga, ON, Canada). cDNA was generated from 1 ug RNA by reverse transcription using the Maxima First-Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) and the following conditions were employed: initial heating at 25 \({}^{\circ}\)C for 10 min, reverse transcription at 50 \({}^{\circ}\)C for 15 min, and enzyme denaturation at 85 \({}^{\circ}\)C for 5 min.  
Gene expression was studied by qPCR using the CFX96 C1000 Touch Thermocyler Real-Time System (Bio-Rad Laboratories Canada, Mississauga, ON, Canada) and SsoFast SYBR Green Supermix (CFX96 C1000 Touch Thermocyler Real-Time System (Bio-Rad Laboratories Canada, Mississauga, ON, Canada)) using Hard Shell PCR 96-well thin walled plates (CFX96 C1000 Touch Thermocyler Real-Time System (Bio-Rad Laboratories Canada, Mississauga, ON, Canada)). PCR reactions were performed in a 10-\(\upmu\)L reaction volume containing 5 \(\upmu\)L SsoFast SYBR Green Supermix, 0.5 \(\upmu\)L each of forward and reverse primer, 1 \(\upmu\)L cDNA, and 3 \(\upmu\)L of RNAse/DNAse free PCR water. PCR reaction conditions included an initial denaturation at 98 \({}^{\circ}\)C for 3 min, followed by 40 cycles of 98 \({}^{\circ}\)C for 10 seconds (s) and 60 \({}^{\circ}\)C for 30 s. The reactions were performed in triplicate and experiments were repeated with three biological replicates. Relative gene expression was determined using the \(\upDelta\)Ct method [38]. The data was analyzed using Cfx96 Manager software version 3.1 (Bio-Rad Laboratories Canada, Mississauga, ON, Canada).