The phnE gene encoding catechol 2,3-dioxygenase belonging to the meta-cleavage pathway was selected as the marker gene and was detected and quantified from soil samples by competitive quantitative PCR. A PCR primer pair was designed based on the phnE gene to amplify the target DNA bands and competitor DNA bands. The phnE gene was detected in two samples of three. In samples S1 and S2, the phnE gene copy number was 6.2 x 10(7)/g soil and 5.8 x 10(7)/g soil, respectively. But no phnE gene was detected in sample S3. The target DNA bands were extracted and expressed. The results confirmed that the target DNA bands were the native phnE genes.
A plasmid pSDK-1 containing the Escherichia coli phosphofructokinase-1 gene (pfkA) was constructed, and transferred into Acidithiobacillus thiooxidans Tt-7 by conjugation. The pfkA gene from E. coli could be expressed in this obligately autotrophic bacterium but the enzyme activity (18 U g-1) was lower than that in E. coli (K12: 86 U g-1; DF1010 carrying plasmid pSDK-1: 97 U g-1). In the presence of glucose, the Tt-7 transconjugant consumed glucose leading to a better growth yield.
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