Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase ( ) g...

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Bibliographic Details
Published in:Bioengineering (Basel) 2017-05, Vol.4 (2), p.52
Main Authors: Montenegro, Eduarda Morgana da Silva, Delabary, Gabriela Scholante, Silva, Marcus Adonai Castro da, Andreote, Fernando Dini, Lima, André Oliveira de Souza
Format: Article
Language:English
Subjects:
Amplification
Bacteria
Biodegradability
Bioengineering
Bioinformatics
Biopolymers
Bioprospecting
Biosynthesis
Colonies
Data banks
Deoxyribonucleic acid
Diagnostic systems
DNA
Enzymes
Exploration
Gene sequencing
Genes
Genetic diversity
Genomes
Genomics
Initiators
Microorganisms
Nucleotide sequence
Organisms
PhaC gene
Polyhydroxyalkanoic acid
Polymerase chain reaction
Polymers
Primers
Screening
Soil sciences
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Summary:The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase ( ) gene, which is involved in the production of polyhydroxyalkanoate (PHA)-a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms ( = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced ( = 4), allowing for the confirmation of the C gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.
ISSN:2306-5354
2306-5354
DOI:10.3390/bioengineering4020052