Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR

Abstract

Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3 % DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1i 10 5 viable cells for Ralstonia eutropha. Nineteen PHA-positive bacteria were used to evaluate this PCR protocol ; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHApositive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible.

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…Although these staining methods are quite sensitive, but it is rather time-consuming and labor-intensive work to screen a large number of environmental isolates. Moreover, prior to identification and isolation of PHA-producing bacteria by phenotypic methods, it is necessary to provide appropriate carbon sources, nutrient limitation conditions and a long culture time is required for PHA granule accumulation to the bacterial cells (78,79). Sudan black B is nonspecific to PHA as it also stains other lipid bodies.…”

“…Genotypic-based screening method is specific, reliable and quick which circumvents the major drawbacks inherent in phenotypic detection methods (78,(81)(82)(83)(84). For genotypic-based screening, the degenerate or and specific oligo nucleotide primers are designed based on multiple sequence alignment results and are used as PCR primers to detect PHA synthase genes (78,79,81,(83)(84)(85). For rapid detection, colony PCR technique is employed for screening PHA producers from the environment (78,80).…”

Biosynthesis of biodegradable polyhydroxyalkanotes biopolymers in genetically modified yeasts

“…To confirm results of the Nile-Red assay, positive clones were screened for the presence of inserts by restriction mapping (Sambrook, et al, 1989). Moreover, PCR detection of PHB genes in the positive yeast clones were performed using PHA specific PCR primers and protocols described elsewhere (Sheu et al, 2000). Based on screening results the transgenic yeasts designated S. cerevisiae INVSc1/PHA and Kloeckera spp KY1/PHA were chosen for further characterization and analysis.…”

COMPARATIVE EVALUATION OF CONVENTIONAL VERSUS RAPID METHODS FOR AMPLIFIABLE GENOMIC DNA ISOLATION OF CULTURED <i>Azospirillum</i> sp. JG3

“…Wan et al (2011) developed a colony PCR procedure with centrifugation, re-suspension, extraction buffer incubation, and heating at 100°C for genetic screening of Chlorella and related microalgae [24]. [14,25]. Unfortunately, all of these cell protocols failed to give satisfying PCR results for our bacterial cells using 20-mer degenerate oligonucleotides.…”

“…Various versions of colony PCR protocols have been developed for microbial cells that are basically differed on the use of centrifugation, extraction buffer, vortex, cell suspension, heating, and cooling [6,[14][15][23][24][25]. Interestingly, changes in these steps highly affect PCR results and this is likely related with the varied nature and components of microbial cell membranes.…”