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Construction of Promoter Probe Vector for Acidithiobacillus Ferrooxidans and Isolation and Characterization of petⅡ Promoter

Author: HuangZhiYing
Tutor: YangYu；WuXueLing
School: Central South University
Course: Biological Engineering
Keywords: Acidophilic Thiobacillus ferrooxidans Promoter probe vector Promoter Site-directed mutagenesis
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 31
Quote: 1
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Abstract


Promoter is important cis-elements in the regulation of gene expression , but also genetic engineering is an important component of the expression vector . Of great significance to the promoter cloning build based engineering vector to express the target protein . In this paper , in order to study prokaryotic promoter structure and function . PSV-β-galactosidase plasmid backbone, by the method of site-directed mutagenesis to introduce a new BstBI single restriction sites , the constructed normal replication and expression in Escherichia coli (Escherichia coli) pSVB promoter probe vector . Acidophilic Thiobacillus thiooxidans (Acidithiobacillus ferrooxidans) consisting of by cycA2, sdrA2, petA282C2 gene pet Ⅱ operon encoding the electron transfer bcl complex located cycA2 upstream sequence of the gene coding region has the structural features of the promoter . This article using the PCR method of the DNA fragment of the gene upstream of the A.ferrooxidans bacteria cycA2 cloned into pSVB promoter probe vector beta - galactosidase gene upstream , instead of its original promoter (gpt promoter) , to give the recombinant plasmid PAP2 . The plasmids were introduced into E. coli (Escherichia coli) expression by detecting β - galactosidase enzyme activity , to identify the strength of the promoter activity . The activity analysis showed that A.ferrooxidans the bacteria cycA2 gene upstream DNA fragment having significant promoter activity , but also shows this promoter probe vector can be used for cloning and identification of important genes from A.ferrooxidans bacteria potential promoter zone. Further sequence analysis revealed that the promoter region of the pet Ⅱ upstream its cycA2 gene coding region - 95bp ~~ - 90bp at ( sequence \sites in the promoter core element , that is similar to the -35 and -10 region sequences . Site-directed mutagenesis techniques , in -95bp ~-90bp at its base by \54bp sequence of the first nucleotide from the \The above results show that : the two regions pet Ⅱ promoter sites identified as the -35 and -10 region of the promoter .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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