Molecular Biology and Genetics

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 10 No. 3, Issue of July 15, 2007
© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received November 20, 2006 / Accepted February 19, 2007
DOI: 10.2225/vol10-issue3-fulltext-13
RESEARCH ARTICLE

A most effective method for selecting a broad range of short and medium-chain-length polyhidroxyalcanoate producing microorganisms

Dolly Margot Revelo Romo
Bioprocesses and Bioprospecting Group
Instituto de Biotecnología de la Universidad Nacional
Ciudad Universitaria, Edificio Manuel Ancizar
Bogotá, Colombia
Tel: 57 1 316 5000. Ext 16970/71
Fax: 57 1 3165415
E-mail: dmrevelor@unal.edu.co

María Victoria Grosso
Bioprocesses and Bioprospecting Group
Instituto de Biotecnología de la Universidad Nacional
Ciudad Universitaria, Edificio Manuel Ancizar
Bogotá, Colombia
Tel: 57 1 316 5000. Ext 16970/71
Fax: 57 1 316 5415
E-mail: mvgrossob@unal.edu.co

Nubia Carmenza Moreno Solano
Bioprocesses and Bioprospecting Group
Instituto de Biotecnología de la Universidad Nacional
Ciudad Universitaria, Edificio Manuel Ancizar
Bogotá, Colombia
Tel: 57 1 316 5000. Ext 16972
Fax: 57 1 3165415
E-mail: ncmorenos@unal.edu.co

Dolly Montoya Castaño*
Bioprocesses and Bioprospecting Group
Instituto de Biotecnología de la Universidad Nacional
Ciudad Universitaria, Edificio Manuel Ancizar
Bogotá, Colombia
Tel: 57 1 316 5000. Ext 16951/52/54
Fax: 57 1 3165415
E-mail: dmontoyac@unal.edu.co

*Corresponding author

Financial support: This research formed part of a project financed by COLCIENCIAS (Instituto Colombiano Francisco José de Caldas) (project 1101-12-12388).

Keywords: 16S rDNA, colony PCR, degenerate primers, PHA synthase, polyhydroxyalcanoate, seminested PCR.

Abbreviations:

16S rDNA: 16S ribosomal gene
ATCC: American Type Culture Collection
bp: base pairs
DMSO: dimethyl sulphoxide
IBUN: Instituto de Biotecnología de la Universidad Nacional
mcl-PHA: medium-chain-length PHAs
OD: optical density
ORF: open reading frame
PCR: polymerase chain reaction
PHA: polyhydroxyalcanoate
phaC: PHA synthase gene
PhaC: PHA synthase
scl-PHA: short-chain-length PHAs

Abstract
Full Text

A molecular approach was used for selecting polyhydroxyalcanoate (PHA)-accumulating potential Gram-negative bacteria from different genera by colony polymerase chain reaction (PCR). Three degenerate primers were designed for amplifying a fragment from PHA synthase gene (phaC) (Class I), phaC1 and phaC2 (Class II) genes for detecting PHA-producing bacteria. Thirty-four out of 55 bacterial strains from the old collection selected using Sudan black B staining were phaC+. PCR was used for directly selecting 35 new collection bacterial strains; these strains were phaC+ and their ability to produce PHA was confirmed by Sudan black B staining. Four specific primers were designed on genes of Class II PHA biosynthesis operon. These primers were used for evaluating 9 strains from the old phaC+ collection; 6 showed Class II PHA synthase organisation. 34 from the old and new bacterial isolation were characterised by 16S ribosomal gene (16S rDNA) gene partial sequencing. The tool proposed here can be used for better directing PHA production based on PHA biosynthesis genes and bacterial genera. Class I or II phaC genes were detected in 9 different genera and were able to infer the type of polymer produced.

Supported by UNESCO / MIRCEN network