Screening And Study Of Acidithiobacillus Caldus Promoters

Acidithiobacillus caldus is an important becterium in bioleaching,which is moderately thermophilic,acidophilic and chemoautotrophic.It can help iron oxide bacteria significantly improve the leaching efficiency in the mixed leaching system.Promoter,is an important cis acting element in the regulation of the gene expression,as well as an essential part of expression vector.The study of promoter is important for constructing gene engineering vectors,reconstructing of metabolic pathways and expressing the target protein.In this paper,based on laboratory existing genomics and transcriptomics data,we predict and analysis the sequence features of gene promoters come from different major metabolic pathways of A.caldus.Promoter-probe plasmids with gusA reporter gene were constructed and transformed into E.coli and A.caldus.we constructed a plasmid containing gusA reporter gene and inducible promoter.Then we measured the expression level of mRNA and the protein specific activity induced by different concentration of IPTG.Then the promoter-probe plasmids were transferred into A.caldus by conjugation and the activity of promoters were detected in A.caldus.The differences of the promoter activity were analyzed.This article is about gene promoters of A.caldus,which made progress in the following four aspects:Firstly,we obtained the upstream sequence of different genes come from c arbon metabolic pathway,nitrogen metabolic pathway and sulfur metabolic path way and so on.By bioinformatic analysis,we obtained the-10 region and-35 region and their score,further,we obtain the distance between them.By anal yzing the promoter sequence,the A.caldus gene promoter-10 region sequence is G33G58G39T73A92T48A42A51T89,and the-35 region sequence is T80T80G74C45C 56A35.Through the analysis of the promoters,the unique characteristics of A.c aldus metabolism would be understood better.Secondly,this is the first report on the mass screening and identification of A.caldus promoters.We mainly verify gene promoters come from sulfur metabolic pathway of A.caldus.Promoter activity was investigated and compared by detecting the gusA activities.The DNA fragments with different promoter activity were obtained,and can be used for the construction of the leaching engineering bacteria.According to the promoter activity we analyse the characteristics of sulfur metabolism pathway.It is found that the analysis results are consistent with the results from earlier laboratory research results.Some highly active promoters are found,and the corresponding gene has not been studied in detail,which lays a foundation for further study of sulfur metabolism pathway and construction of genetically engineered bacteria.Thirdly,we constructed a plasmid containing gusA reporter gene and inducible promoter.Then we measured the expression level of mRNA and the protein specific activity induced by different concentration of IPTG and find that the transcription level and the translation level have a very good consistency.Fourthly,promoter probe plasmid are transferred into A.caldus by conjugal with E.coli.According to the expression level of mRNA,we found that the difference of different promoter activity is getting smaller.,but the overall trend of them is similar.Only one of the six promoter activity was significantly decreased,which may be related to the differences in the genetic background of A.caldus and E.coli.