Construction method and application of bacterial promoter reporter vector

A construction method and promoter technology, applied in the biological field, can solve the problems of unstable fluorescent quantitative PCR results, expensive fluorescent dyes, and high cost, and achieve stable and reliable results, low cost, and strong inclusiveness

Inactive Publication Date: 2019-12-31
武汉博欧特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because RNA is unstable and easily degraded, the efficiency of reverse transcription fluctuates, and the price of

Method used

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  • Construction method and application of bacterial promoter reporter vector
  • Construction method and application of bacterial promoter reporter vector
  • Construction method and application of bacterial promoter reporter vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the construction of reporter carrier

[0033] S1. Construction of reporter vector pBBRMCS-lacZ

[0034] The schematic diagram of the construction of the reporter vector pBBRMCS-lacZ is as follows: figure 1 As shown, using the primer pair P1 / P2, using the pBBR1MCS-5 plasmid as a template, the plasmid backbone fragment was obtained by reverse amplification, including the gentamicin resistance gene, replicon and mob transfer gene; using the primer pair P3 / P4, The lacZ gene was amplified using the pSH18-34 plasmid as a template; the above two PCR products were digested with KpnI and XbaI, ligated with T4 DNA ligase, and verified by sequencing to obtain the vector pBBRMCS-lacZ. Primer P3 has recognition sequences of 7 common restriction endonucleases (KpnI, XhoI, HindIII, EcoRI, PstI, SmaI and BamHI), which provide multiple cloning sites for the vector after ligation. Such as figure 1 In the reporter vector pBBRMCS-lacZ shown, the tac promoter and lacI const...

Embodiment 2

[0049] Embodiment 2, detecting the promoter activity of fleS gene

[0050] S1. Construction of the reporter vector pBBRMCS-fleSpro-lacZ

[0051] The map of the reporter vector pBBRMCS-fleSpro-lacZ is as follows image 3 shown. FleS is a histidine kinase in Pseudomonas, and its gene promoter activity is positively regulated by FleQ, and the loss of fleQ will lead to a decrease in fleS promoter activity. The present invention uses the fleS promoter as the target promoter to test the feasibility of the present invention. Using the total genomic DNA of Pseudomonas putida KT2440 as a template, the promoter of the fleS gene was amplified using the primer pair P5 / P6, and then ligated into the pBBRMCS-lacZ vector with BamHI and EcoRI, and sequenced to verify that the promoter sequence was correct. The reporter vector pBBRMCS-fleSpro-lacZ was obtained.

[0052] S2. Detecting the activity of the fleS promoter

[0053] The pBBRMCS-fleSpro-lacZ vector was introduced into the wild-typ...

Embodiment 3

[0054] Example 3, detection of whether the unknown DNA fragment has promoter activity

[0055] S1. Construction of reporter vectors pBBRMCS-PP_1493pro-lacZ and pBBRMCS-PP_1494pro-lacZ

[0056] PP_1493 and PP_1494 are two genes in the wsp operon of Pseudomonas putida KT2240. PP_1494 is responsible for the synthesis of the second messenger c-di-GMP, and PP_1493 inhibits the synthesis activity of PP_1494. The two genes are closely related to the formation of biofilm . Whether the sequences in front of the PP_1493 and PP_1494 genes have promoter activity is still unclear. The present invention detects whether an unknown sequence upstream of the PP_1493 and PP_1494 genes has promoter activity to test the feasibility of the present invention. Use the primer pair P7 / P8 to amplify the upstream 500bp sequence of the PP_1493 gene, digest it with EcoRI+BamHI, and connect it to the pBBRMCS-lacZ vector to obtain pBBRMCS-PP_1493pro-lacZ. The plasmid map is as follows Figure 5 shown. Use...

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Abstract

The invention discloses a construction method and application of a bacterial promoter reporter vector. The method includes the following steps: first constructing a vector containing beta-galactosidase lacZ gene, inserting a target gene promoter into the upstream of the lacZ gene in the vector so as to obtain a reporter vector for detecting the activity of the gene promoter, introducing the reporter vector into to-be-tested strains, and inferring the change of the activity of the target gene promoter by detecting and comparing the activity of the strains and the beta-galactosidase. The reporter vector of the present invention can be used to detect the activity of gene promoters in bacteria and determine the strength of gene expression, and can also be used to detect the presence or absenceof promoter activity of unknown DNA fragments in bacteria, and to determine the location of the gene promoters with low operation cost and stable and reliable result.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a bacterial promoter reporter vector and its application. Background technique [0002] Transcription and translation are continuous in prokaryotes, so regulation at the transcriptional level is the most important regulation in prokaryotes. The transcriptional expression of a gene requires a promoter, and the activity of the promoter determines the expression level of the gene. Detection and comparison of gene promoter activity is of great significance for understanding the function and regulation of genes. At present, the commonly used method for detecting gene promoter activity is fluorescent quantitative PCR. The general steps are to extract total RNA, reverse transcribe to obtain cDNA, and use a PCR system with fluorescent dyes to quantify the amount of cDNA to evaluate the amount of mRNA transcribed from the target gene in the total RNA to compare the...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/56C12Q1/34
CPCC12N9/2471C12N15/66C12Q1/34C12Y302/01023G01N2333/938
Inventor 崔格特
Owner 武汉博欧特生物科技有限公司
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