Detection of the enzymatically-active polyhydroxyalkanoate synthase subunit gene, phaC, in cyanobacteria via colony PCR

• Published in: Molecular and cellular probes Vol. 29; no. 6; pp. 454 - 460
• Main Authors: Lane, Courtney E., Benton, Michael G.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2015
• Subjects: Acyltransferases - genetics; Acyltransferases - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Colony PCR; Cyanobacteria; Cyanobacteria - enzymology; Cyanobacteria - genetics; Cyanobacteria - growth & development; DNA Primers - genetics; PCR; PHA; PHA synthase; Polyhydroxyalkanoate; Polyhydroxyalkanoates - biosynthesis; Polymerase Chain Reaction - methods; Sequence Analysis, DNA - methods; PHA; Colony PCR; PHA synthase; PCR; Polyhydroxyalkanoate; Cyanobacteria
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2015.07.001

A colony PCR-based assay was developed to rapidly determine if a cyanobacterium of interest contains the requisite genetic material, the PHA synthase PhaC subunit, to produce polyhydroxyalkanoates (PHAs). The test is both high throughput and robust, owing to an extensive sequence analysis of cyanobacteria PHA synthases. The assay uses a single detection primer set and a single reaction condition across multiple cyanobacteria strains to produce an easily detectable positive result – amplification via PCR as evidenced by a band in electrophoresis. In order to demonstrate the potential of the presence of phaC as an indicator of a cyanobacteria's PHA accumulation capabilities, the ability to produce PHA was assessed for five cyanobacteria with a traditional in vivo PHA granule staining using an oxazine dye. The confirmed in vivo staining results were then compared to the PCR-based assay results and found to be in agreement. The colony PCR assay was capable of successfully detecting the phaC gene in all six of the diverse cyanobacteria tested which possessed the gene, while exhibiting no undesired product formation across the nine total cyanobacteria strains tested. The colony PCR quick prep provides sufficient usable DNA template such that this assay could be readily expanded to assess multiple genes of interest simultaneously. •We analyze 29 cyanobacteria polyhydroxyalkanoate synthase sequence accessions.•Consensus degenerate PCR primers are designed based on our analysis.•PCR amplification is performed using primers designed.•We compare PCR amplification results to traditional screening methods.•A standardized high-throughput PCR assay is developed and tested on nine cyanobacteria.