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A technology of Streptomyces coelicolor and bidirectional promoter, which is applied in the field of genetic engineering, can solve the problem that the unidirectional promoter is difficult to achieve, and achieve the effect of improving the expression efficiency
Inactive Publication Date: 2019-02-01
TIANJIN UNIV
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However, when two or more genes are introduced, the expression level and expression time of these genes are usually required to be identical, so as to ensure that the transferred genes can perform normal functions. It is difficult to meet such requirements by using a unidirectional promoter.
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Embodiment 1
[0023] Example 1: Construction of a Streptomyces coelicolor bidirectional promoter SCP1 (hereinafter referred to as SCP1) unilaterally connected to the green fluorescent protein gene (eGFP, referred to as the reporter gene) recombinant
[0025] (2) Using genomic DNA as a template and using SCP1-F and SCP1-R as primers for PCR amplification, the reaction system is 50ul, including ddH2O 18ul, dNTP mixture 1ul, SCP1-F primer 1ul, SCP1-R primer 1ul, DNA Template 1ul, 2×Phanta Max 25ul, Fidelity DNA polymerase 1ul.
[0026] The amplification process is: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing at 66°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 5min
[0027] Primers used:
[0028] SCP1-F 5'GCGCCACCCCTCGACACAG 3' (SEQ ID NO. 2);
[0029] SCP1-R 5'GCGCCACCCCTCGACACAG 3' (SEQ ID NO.3)
[0030] (3) The PCR product wa...
Embodiment 2
[0058] Example 2. Construction of SCP1 promoter bilaterally linked reporter gene recombinant
[0060] (2) Using genomic DNA as a template and using SCP1-F and SCP1-R as primers for PCR amplification, the reaction system is 50ul, including ddH2O 18ul, dNTP mixture 1ul, SCP1-F primer 1ul, SCP1-R primer 1ul, DNA Template 1ul, 2×Phanta Max 25ul, Fidelity DNA polymerase 1ul.
[0061] The amplification process is: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing at 66°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 5min
[0062] The primers used were: SCP1-F 5'GCGCCACCCCTCGACACAG 3'; SCP1-R 5'GCGCCACCCCTCGACACAG 3'
[0063] (3) The PCR product was detected by agarose gel with a mass volume ratio of 1.5%, and the target fragment was recovered with a gel recovery kit.
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Abstract
The present invention discloses a streptomyces coelicolor bidirectional promoter SCP1, and a nucleotide sequence of the bidirectional promoter SCP1 is shown as SEQ ID NO. 1; or the nucleotide sequencecomplementary to the nucleotide sequence shown in SEQ ID NO.1. When constructing a geneexpression vector, a promoter sequence is added while plasmid designing to ensure expression of a target gene.The bidirectional promoter is placed between the two ORF to express both ORF simultaneously. Since the two ORF have own start codon and stop codon, two independent and unmodified proteins are translated, the problems of mutual interference or inhibition of the promoters in a multi-promoter carrier cannot be generated, so that theeffect of efficient expression of the target gene can be achieved. Norelated report on the bidirectional promoter in streptomyces coelicolor is generated, the present invention provides the streptomyces coelicolor bidirectional promoter SCP1, which can greatly improvethe gene expression efficiency.
Description
technical field [0001] The invention relates to the field of genetic engineering, in particular to a Streptomyces coelicolor bidirectional promoter SCP1 and a Streptomyces coelicolor bidirectional promoter SCP1 recombinant vector containing the promoter. Background technique [0002] Promoter refers to a DNA sequence located upstream of the genetranscription initiation site, capable of binding to RNApolymerase and other transcription factors, and then regulating the transcription initiation and transcription efficiency of the downstream target gene. Promoters have important application value in designing and preparing genetic engineering products. When the distance between the adjacent transcription start sites (transcription start sites, TSS) of two genes transcribed in opposite directions is less than about 1 kb, this intergenic DNA sequence can be called a "bidirectional promoter". )". Bidirectional promoters belong to a special class of promoters capable of simultane...
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