|554-1||Colony PCR selects potentially bacteria-producing soil polyhydroxyalcanoates adhered to the surface yam tuber|
|Autores:||Juliana de Castro Nunes Pereira (UFPE - CAV - Universidade Federal de Pernambuco - Campus Vitória) ; Rafaela Azevedo Abrantes de Oliveira (UFPE - CAV - Universidade Federal de Pernambuco - Campus Vitória) ; Romero Marinho de Moura (UFPE - CAV - Universidade Federal de Pernambuco - Campus Vitória) ; Christine Luna Finkler (UFPE - CAV - Universidade Federal de Pernambuco - Campus Vitória) ; Leandro Luna Finkler (UFPE - CAV - Universidade Federal de Pernambuco - Campus Vitória) ; Idjane Santana de Oliveira (UFPE - CAV - Universidade Federal de Pernambuco - Campus Vitória) |
The Yam is a plantation of social and economic importance to the Brazilian Northeast, being rich in vitamins and food starch. Polyhydroxyalcanoates (PHAS) are biodegradable polymers used as raw material for the manufacture of plastics and are produced by bacteria also from starch. The goal of this study was to isolate the ground joined the surface of yam tuber potentially bacteria-producing PHAS, selecting them by PCR of the colony, from gene amplification phaC that encodes the enzyme PHA synthase of PHA synthesis pathway. Positive PCR colonies were confirmed with staining with sudan black for subsequent production of PHAS by fermentation in bioreactor systems. Were collected two yam tubers with soil adhering to surface in commercial planting area in Pernambuco, Brazil. Serial dilutions were made of soil samples (after scaling) in saline solution up to 10-9 plated in nutrient agar culture medium and incubated for 48 hours at 37oC. Approximately sixteen bacterial colonies with distinct morphological characteristics were used for colony PCR. The DNA of the colony was extracted by boiling method using 50 µl of TE and a strap of the colony removed from the plate. It was then held colony PCR using primers for the gene phaC (GD and DR that amplify DNA fragment of 551pb), in a PCR reaction mix (25 µl) containing 0.5 µM dNTPs mix, 1. 25 mM MgCl2, 0.3 µM of each primer and 0.25UI of Taq and 2 µl of DNA extracted from the colony. The amplified DNAs were separated in gel electrophoresis (1.0%), stained with syber safe and displayed in blue light transilluminator. Were used as positive control of PCR, the DNA of bacteria used in the laboratory for the production of PHAS, Cupriavidus necator, Pseudomonas putida and Bacillus thurigiensis israelensis (Bti) and negative control PCR mix without DNA. Of the 16 colonies, 5 (31.2%) showed strong sudan staining black and were positive for the gene PCR phaC. Among the positive bacterial colonies in PCR were found a coccus gram positive bacteria, two gram negative coccus and two gram-positive bacilli. The associated colony PCR the coloration of the colony with sudan black proved to be efficient and quick to check potentially bacteria-producing PHAs, being the test carried out in one day.
Palavras-chave: bacteria, biopolymer, PCR, soil