The bacterial strains used were E.coli BL(DE3) and Pseudomonas citranolleise (P.citranolleise) (DSMZ 11735). E.coli was grown at 37°C in Luria-Bertani medium (LB). P.citranolleise was grown in Minimal Salt Medium (MSM) containing 1.5% glucose. When needed, ampicillin and Isopropyl-b-D thiogalactopyranoside (IPTG) were added to the medium.

Two plasmids, pTZPHAC1 and pTZPHAC2, which contained the PHA synthase genes (phaC1 and phaC2) from P. aeruginosa PTCC 1310, were constructed from genomic DNA as described previously. (9) These plasmids were extracted and digested with restriction enzymes. The NdeI and BamHI enzymes were used to digest pTZPHAC1 and for digestion of pTZPHAC2 NdeI was used (BamHI was not used because it digests the phaC2 sequence). The obtained phaC1 and phaC2 inserts were ligated into pET15b (Novagen, USA) expression vector digested with NdeI, BamHI and NdeI, respectively. These recombinant vectors were transformed into competent E.coli BL21 (DE3) cells using the CaCl₂ method (10).

For the expression of PHA synthase, a positive cell clone was inoculated into 5 ml of LB medium (100 µg/ml Amp). An overnight culture was used to inoculate (1% v/v) 50 ml of fresh LB medium in 250 ml flasks and incubated at 37°C to an OD_600nm of 0.4-0.6. Then the expression of phaC genes was induced by the addition of IPTG to a final concentration of 1 mM. The expressed enzymes were purified with Ni-NTA His Bind Resin (Novagen, USA) as described by the manufac-turer.

Briefly, 1 ml of each sample was collected and centrifuged after an appropriate growth period. The pellets were resuspended in 200 µl of buffer A and clarified by centrifugation at 15000 g for 10 min and the supernatant was taken for the next steps. Fifty µl of the 50% 1X Ni-NTA His Bind Resin was added to each sample and mixed gently for 30 min at room temperature. After centrifugation of samples, the supernatant was removed and the pellet was washed two times with 250 µl of buffer C. Finally, the His-tagged enzyme was eluted with buffer E and 20 µl of each sample was electrophoresed on a 12% polyacrylamide gel using SDS-PAGE.

IPTG concentration, time and temperature of incubation with IPTG were optimized for phaC genes expression in E.coli BL21 (DE3) cells. For optimization of enzyme expression, IPTG concentration at four levels (0.5, 1, 1.5 and 2 mM), three different times of incubation with IPTG (1, 2 and 3 hrs) and the temperature of incubation with IPTG at three levels (25, 37 and 39°C) were examined. Each assay was repeated three times for pET-phaC1 and pET-phaC2 and one time for pET15b as the negative control (a total of 252 experiments). Expressed protein samples were analyzed on a 12% SDS-PAGE gel and the intensity of bands were quantified and compared using SPSS 10 version and differences at p<0.05 were considered as significant.

To confirm the expression of functional PHA synthase enzymes by E.coli BL21 (DE3) cells car-rying the phaC genes, the accumulation of PHA in bacteria was detected with Nile red (11). The E.coli BL21 (DE3) cells carrying the phaC gene, E.coli BL21 (DE3) without the phaC gene (as the nega-tive control) and P.citranolleise (as the positive control) were investigated for PHA production. E.coli BL21 (DE3) cells were streaked on an LB plate supplemented with sodium-gluconate (0.5% w/v), Nile red (0.05 µg/ml), IPTG (0.1 mM) and ampicillin (100 µg/ml). P.citranolleise was grown on MSM supplemented with sodium-gluconate (0.5% w/v), glucose (1.5% w/v) and Nile red (0.05 µg/ml). The agar plates were incubated at 37°C for 24 hr.

The PHA synthase enzymes were expressed by the addition of IPTG, then the cell suspension was centrifuged. The pellets were added to Ni-NTA His Bind resins. After purification, samples were analyzed by SDS-PAGE, which revealed that the target protein of 62 kD was expressed (Figure 1).

The effect of incubation temperature and IPTG concentration on SDS-PAGE band intensity of phaC1 and phaC2 were investigated and the results are shown in Figure 2. The maximum production of the PHA synthase enzymes occurred when 1 mM IPTG was used at 37°C. The effects of incubation time and IPTG concentration on SDS-PAGE band intensity of phaC1and phaC2 were evaluated (Figure 3). Maximum production of PHA synthase enzyme was observed when 1 mM IPTG was used for 2 hr.

Nile red plate assay revealed functional expression of PHA synthase by recombinant E.coli BL21 (DE3) cells (Figure 4).