A Promoter Probe Vector And Low Temperature-induced Promoter

Promoter, an essential part in the vector for gene engineering expression, is an important element in the regulation of the gene expression. The cloning technology of promoters includes screening promoters with probe carriers and cloning promoters with PCR technology.Green fluorescent protein (GFP) was discovered from Aequorea Victoria,it can emit green light under excitation of blue or UV irradiation. GFP as a marker for the gene expression and localization of gene products has been widely used in life sciences for the past years because of its stable structure and photo physicalproperty and easy expression in cells. In this paper, plasmid pAcGFP was used for construction of a green fluorescent protein promoter-probe vector. A fragment of 700bp gene has been ligased into the pAcGFP vector plasmids instead of the lac promoter, resulted in the mediate plasmid pEH+GFP. A pair of special designed DNA fragment, RBS,was inserted into pEH+GFP to construct the plasmid pEH+GFP+RBS.Finally, a GFP promoter-probe vector, pGFP+RBS,was constructed and used successfully for the identification of a constitutive promoter from DNA of P.Aeruginos.CspA, the major cold-shock protein of Escherichia coli, has recently been studied with respect to its structure, function and regulation at the level of transcription, translation and mRNA stability. We cloned the promoter of the cspA gene by PCR technology to develop a low temperature induced expression vector. The CspA promoter gene fragment was obtained by PCR using a pair of special designed primers from the DNA of Escherichia coli. A fragment of T7 promoter in pET30a(+)was replaced by the CspA promoter(cold promoter) to construct a low temperature-induced expression vector pET30a/cold. The function of pET30a/cold was determined by expression the gene of arginine deiminase (ADI). SDS-PAGE assay result of ADI showed pET30a/cold-ADI expression efficiency was identical to pET30a-ADI. The results indicated that the cloned promoter fragment contains the complete center promoter of the cold shock protein CspA. The promoter fragment was a strong promoter with high activity in E.coli The experiments suggested that this low temperature induced expression vector pET30a/cold could be used for protein over expression in low temperature. This new expression vector as an engineering tool could be used to lay the groundwork for future research.