Genetic Transformation of Amylase Gene to Ruminal <i>Bacteroides</i> Species Using Conjugation Consequence for Improvement of Rumen Enzyme

Abstract

Rumen bacterial strains can potentially be manipulated to perform functions different from wild type species. The most numerous species of bacteria in the rumen and gut are species of the familyBacteroidetes, whichcan have the potential for genetic modification for enzyme production. One of the genetic manipulation of rumen bacteria can perform for production of starch digestive enzyme for the enhancement of nutrient flow to the rumen. In this study, <em>Bacteroides </em>species were isolated from rumen of cows. The 16S rRNA gene analysis was used to confirm the classification of the <em>Bacteroides </em>species. The amylase gene from <em>Bacillus </em>spp. was cloned into expression vector pET28A and then subcloned in <em>Escherichia coli</em>-<em>Bacteroides</em> pGFK114.1 shuttle vector. Conjugation between <em>Escherichia coli</em> and a <em>Bacteroides </em>strainwas accomplished with two <em>Escherichia coli</em> donors containing pGFK114.1 and pRK231 and one <em>Bacteroides </em>strain as a recipient. Transfer of amylase gene by vector was confirmed using a marker cefoxcitin antibiotic resistant gene. The amylase activity assay showed that successful expression of enzyme occurred in ruminal <em>Bacteroides </em>species.