Plasmids, bacteria and chemicals
The plasmid pPhaCAB consists of a pBluescript II SK+ backbone (Stratagene, USA) and the PHB biosynthetic gene cluster encoding three genes for type I PHA synthase (phaC), ketothiolase (phaA), and acetoacetyl-CoA reductase (phaB) from Cupriavidus necator H16 (Yang et al. 2010). Escherichia coli XL1-Blue (Stratagene) was transformed with pPhaCAB for expression of the PHA biosynthesis genes. LipidGreen1 was provided by Korea Chemical Bank (KRICT, South Korea; Additional file 1: Fig. S1). LipidGreen1and Nile red stock solutions were prepared by dissolving the dyes in dimethylsulfoxide (DMSO) to a final concentration of 1 mg/mL. PHB powder was purchased from Sigma-Aldrich (USA). Ten milligrams PHB powder was suspended in 1 mL water using ultrasonic homogenizer (Sonics and Materials, USA) for 1 min on 20% amplitude.
Recombinant E. coli XL1-Blue transformed with phaCAB, PHB-producing cell, was grown at 37°C in Luria–Bertani (LB) medium containing 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, and 50 μg/mL ampicillin. After 20 h cultivation in 2 mL of LB broth, the PHB-producing cells were inoculated into LB medium supplemented with 20 g/L glucose and cultured on an incubator at 37°C for 20 h with shaking (200 rpm). For cell viability analysis, the PHB-producing cells were cultivated in 100 mL LB medium with 20 g/L glucose and LipidGreen1 (0, 0.8, and 2 µg/mL). The cultures were collected every 2 or 3 h and then optical densities at 600 nm were measured (Shimadzu, Japan).
Observation of bacterial PHB on an agar plate
The PHB-producing cells were spread on the agar plate containing LipidGreen1 at a final concentration of 25 µg/mL and cultured for 20 h at 37°C. Accumulation of intracellular PHB was viewed under ultraviolet light (302 nm). E. coli XL1-Blue, which contains only the pBluescript II SK+ vector (Agilent Technologies, USA), was prepared as a negative control. Subsequently, the PHB-producing and PHB-non-producing cells were scraped from the surface of the agar plates and suspended in 100 µL of phosphate-buffered saline (PBS, pH 7.2, 20 mM). Ten microliters of the suspensions were placed on slide glass and used for microscopic observation by fluorescence microscope (Nikon, Japan) with a green fluorescence filter (Green Excitation 460–500 nm, Emission 510–560 mm).
Measurement of the fluorescence intensity
The PHB-producing cells were cultivated in 500 mL LB medium containing 20 g/L glucose. The cells were harvested by centrifugation (3,200×g, 4°C for 10 min) and resuspended in PBS to yield an optical density at 600 nm of 2.0. LipidGreen1 was added to the 1 mL cell suspensions, followed by further incubation for 0.5 and 2 h in the dark. The final concentration of LipidGreen1 at 2 µg/mL was used in further experiment. One hundred microliters of the suspensions were immediately transferred into a 96 well black microplate, and the fluorescence intensity was measured within 10 min with a micro-fluorospectrometer (TECAN, Switzerland) at an excitation wavelength of 450 nm and emission wavelength of 510 nm. To verify the relation between the PHB accumulation contents and fluorescence intensity, 30 mL of the culture solutions were collected at 2 h intervals during cell growth and stored at −70°C deep freezer for fluorescence and GC analysis. In addition, the aqueous PHB suspension was serially diluted in water and then incubated with LipidGreen1 for 30 min in black microtubes followed by measurement of fluorescence intensities. Furthermore, Nile red was added to the 1 mL cell suspensions to give a final concentration of 2 µg/mL, and then fluorescence intensities were measured at 540 and 570 nm for the excitation and emission wavelengths, respectively.
Comparison of fluorescence intensity between intact and lysed cells
The PHB-producing cells grown in the 50 mL medium were collected by centrifugation (4°C at 3,200×g for 10 min) to an optical density at 600 of 4.0. Half of the cell suspension in PBS buffer was disrupted with the ultrasonic homogenizer, while the remaining suspension was left on ice as intact cells. One milliliter each of disrupted and intact cell suspension was moved into a black microtube followed by the addition of LipidGreen1. The mixtures were incubated for 0.5 and 2 h and immediately transferred into a black 96-well microplate to measure the fluorescence intensity.
PHB quantification by gas chromatography (GC)
The PHB polymer content was determined by GC analysis as previously described (Yang et al. 2010). Briefly, PHB-producing cells were washed twice with PBS buffer and dried at 65°C in an oven with a final dry pellet weight of 0.03 g. The dry matter was subjected to methanolysis in the presence of 1 mL PHA solution containing 0.8% (wt/vol) Benzoic acid, 3% (vol/vol) sulfuric acid, 97% (vol/vol) methanol and 2 mL of chloroform. Following 6 h of incubation at 100°C, the polymer solutions dissolved in chloroform were precipitated and separated with chilled deionized water. The PHB contents were analyzed by GC (6890N GC system, Agilent Technologies) equipped with a fused silica capillary column (SPBTM-5, 30 m × 0.32 mm ID, 0.25 µm film; Supelco, USA) using benzoic acid as an internal standard.
Construction and screening of a phaC mutant library
Random mutagenesis was performed by error-prone PCR with GeneMorph II Random mutagenesis kit (Stratagene) following the manufacturer’s instructions. Briefly, to introduce random mutations into the phaC gene, forward and reverse primers (ReCMutF; 5′-GATCCCCCGGGCAAGTACC-3, ReCMutR; 5′-GGGAACCTGCAGGCCTGC-3′) were designed based on the nucleotide sequences outside of the structural gene. Each PCR contained 30 ng of the pPhaCAB plasmid as the initial template, 250 ng of each primer, 200 μM of each dNTP, and 5 U of Taq DNA polymerase in Taq DNA polymerase reaction buffer. The PCR started with a denaturation step at 95°C for 30 s, followed by 25 cycles of amplification (30 s at 95°C, 30 s at 55°C, and 3 min at 72°C), and a final extension step at 72°C for 10 min. The PCR products purified with the QIAquick PCR Purification Kit (Qiagen, USA) were digested with SmaI and SbfI and subjected to preparative electrophoresis in a 0.8% agarose gel. The approximately 1.8-kb PCR fragments were ligated into the same restriction sites of the pPhaCAB vector, and then the ligates were transformed into E. coli XL1-Blue. Each mutant clone was grown in a deep-well microplate containing 800 µL of the LB medium containing 20 g/L glucose for 20 h, of which 100 µL were transferred to a black microplate to measure its fluorescence intensity by adding LipidGreen1. Determination of the cellular PHB content by GC was done with selected clones that showed relatively higher or lower fluorescence intensities than that of the wild type.