## 2 Materials and Methods
### Construction of P. putida LS461 with Partial Deletion of the PHA Synthesis Gene Cluster  
The PHA synthesis gene operon of _P. putida_ LS46 was identified from the complete genome sequence ([[http://www.ncbi.nlm.nih.gov/nucore/ALPV0200008](http://www.ncbi.nlm.nih.gov/nucore/ALPV0200008)]([http://www.ncbi.nlm.nih.gov/nucore/ALPV0200008](http://www.ncbi.nlm.nih.gov/nucore/ALPV0200008))) coordinate 202310-197359; 13) and encoded the following genes: _phaC1 phaZphaC2 phaD_. A deletion mutant, designated _P. putida_ LS461, was constructed by deleting the 3_'-phaC1,phaZ_ and 5_'-phaC2_ genes from the _P. putida_ LS46 _pha_ operon. Primers were designed for the left and right flanking regions of the _phaC1 phaZphaC2_ genes (PPUTLS46_005625, 1683 bp; PPUTLS46_005620, 852 bp; and PPUTLS46_005611, 1680 bp) (Table 1). The size of nucleotide sequence spanning from _phaC1_ to _phaC2_ was 4325 bp. A partial sequence of _phaC1_ (840 bp, from 426 to 1265 nt) was amplified with Primer 1 (Pphac1 forXbal), which contained an Xbal restriction endonuclease site, and Primer 2 (Pphac1 revHindIII), which contained a HindIII restriction endonuclease site. The amplified fragment was digested with restriction enzymes Xbal and HindIII and ligated into Xbal-HindIII digested pK18 mobascB vector [32], resulting in plasmid pHAC1. Another fragment of 857 bp (from 2950 to 3807 nt) of the _phaC2_ gene was amplified with Primer 3 (Pphac2 forHindIII), which contained a HindIII site, and Primer 4 (Pphac2 revNhel), which contained an Nhel site. The amplified fragment was digested with HindIII and Nhel, and ligated into HindIII-Nhel digested pPHAC1 to form pPHAC1C2. Plasmid pPHAC1C2 had a deletion of the _3'-phaC1_ gene, complete _phaZ_ gene, and 5_'-phaC2_ gene. The plasmid pHAC1C2 was a narrow host range plasmid with deletion in the _phaC1 phaZphaC2_ genes. This plasmid was transferred to _P. putida_ LS46 by triparental mating and transconjugants were selected on LB plates containing chloramphenicol (200 ug/mL) and kanamycin (50 ug/mL) [31]. Two kanamycin-resistant clones were selected and grown in 10 mL LB broth at 30 \({}^{\circ}\)C for 18 h. \begin{table}
\begin{tabular}{c l l l} \hline \hline
**Strain/Plasmid/Primer** & \multicolumn{1}{c}{**Characteristics**} & **References** \\ \hline _P. putida_ LS46 & _P. putida_ isolated from waste water & [13] \\ \hline _P. putida_ LS461 & _phaC1-phaZ-phC2_ deletion mutant of _P. putida_ LS46 & This study \\ \hline _P. putida_ LS46123 & _P. putida_ LS461 carrying plasmid pJC123; \(\mathrm{\text{T}^{R}}\) & This study \\ \hline _E. coli_ DHT5\(\alpha\) & \(\Delta\)_lacZ_, \(\Delta\)15, \(\Delta\)(_lacZYA-argF_), U169, _recAI_, _endAI_, & Qiagen, Hilden, Germany \\ \hline pRK7813 & IneP oriT cos Los2_lac_, \(\mathrm{\text{T}^{R}}\) & [30] \\ \hline pJC123 & Derivative of pRK813 carrying _phaC1\({}_{R}\)_ gene from clone 16; \(\mathrm{\text{T}^{R}}\) & [31] \\ \hline pRK18 mobascB\(\delta\) & Narrow host-range doing vector; _sacB_, \(\mathrm{\text{K}m^{R}}\) & [32] \\ \hline pRK2013 & Hepler plasmid pRK290 derivative; \(\mathrm{\text{K}m^{R}}\) & [33] \\ \hline pPHAC1C2 & pK18 mobascB carrying 840 bp from _phaC1_ and 857 bp from _phaC2_ & This study \\ \hline pPhaC1 forXbal & \(\mathrm{\text{9}^{\circ}}\)CTAGCTCTAGATGCGCGTCAAAACCTTCTTCGAAAC-3\({}^{\prime}\) & This study \\ \hline PPhac1 revHindIII & \(\mathrm{\text{9}^{\circ}}\)GATCAAGCTGTCACGCGGTCCTTGAAACATT-3\({}^{\prime}\) & This study \\ \hline PPhac2 forHindIII & \(\mathrm{\text{9}^{\circ}}\)GATCAAGCTTAGACACCCCAGATGATGAAA-3\({}^{\prime}\) & This study \\ \hline PPhac2 revNhel & \(\mathrm{\text{9}^{\circ}}\)CTAGGCATGCTTCCGAGCAGTGTTG-3\({}^{\prime}\) & This study \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Bacterial strains, plasmids, cosmids, and primers used in this study. Restriction sites are underlined. Serial dilutions of the culture were spread on LB plates with 5% (\(w/v\)) sucrose. Clones were screened for the loss of the kanamycin marker. One kanamycin-sensitive clone obtained in this manner (_P.