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Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof

A technology of Salmonella typhi and Salmonella, applied in the direction of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effects of enhanced fluorescence intensity, sensitive detection, and improved sensitivity

Inactive Publication Date: 2012-01-18
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0003] The traditional luminescent bacteria method can only give the comprehensive toxicity of the sample, while the urban sewage and industrial wastewater with increasingly complex components require daily environmental monitoring not only to reflect the total toxicity of the water body, but also to provide information on different types of toxic substances

Method used

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  • Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof
  • Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof
  • Transgenic salmonella typhimurium TA1535/Pcda-GFP and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0021] 1. Amplification of strong promoter Pcda:

[0022] Using the plasmid containing the Pcda promoter provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd. (the product name is Pcda) as a template, using C1 and C2 as upstream and downstream primers respectively, the PCR reaction system is (Pcda promoter plasmid 100ng, 1μL; C150μM, 1μL; C250μM, 1μL; dNTP (10mM) 1μL; Taq enzyme 5-10U, 1μL; low-salt buffer, 5μL; deionized water, 45μL), carry out PCR reaction (95℃4min, 95℃45s, 52℃ 30s, 72°C 30s, 29cycle, 72°C 10min, 22°C forever), the PCR product was electrophoresed with 1% agarose, and the size of the amplified fragment was shown to be about 120bp ( figure 1 ), which is consistent with the literature reports, and the results are in line with expectations.

[0023] C1:N:5′-AGCGAATTCGGGTTGACAGGATTTACG-3′

[0024] C2:C:5′-TTTCTCCTTTACTCATGCAAACACCT-3′

[0025] 2. Amplification of the green fluorescent protein gene EGFP:

[0026] Using the plasmid containing ...

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Abstract

The present invention discloses transgenic salmonella typhimurium TA1535 / Pcda-GFP and a construction method thereof. The preservation number of the transgenic salmonella typhimurium TA1535 / Pcda-GFP is CCTCC NO: M 2011269. According to the construction method, recombinant plasmid with a strong promoter Pcda and green fluorescent protein gene EGFP is transformed into salmonella typhimurium TA1535 to obtain the transgenic salmonella typhimurium TA1535 / Pcda-GFP. Compared to the wild type salmonella typhimurium TA1535, the engineered strain provided by the present invention provides enhanced fluorescence intensity, thus, when the engineered strain is adopted for a toxicity test, the sensitivity of the toxicity test method is improved, the toxic material can be detected conveniently and rapidly. Therefore, the transgenic salmonella typhimurium TA1535 / Pcda-GFP provided by the present invention provides convenience for realization of convenient, rapid and sensitive detection of the toxic material, and can be applicable for popularization and application in detection of the genetic material in the environment.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a transgenic Salmonella typhimurium TA1535 / Pcda-GFP engineering strain and a construction method thereof. Background technique: [0002] Traditional toxicity test methods, such as fish test, algae test, zooplankton test, etc., are relatively expensive, or the operation is cumbersome and time-consuming, which is not conducive to rapid detection or online monitoring. The luminescent bacteria method can overcome these defects well, and it has the advantages of rapidity, sensitivity and simple operation. Toxicity testing using luminescent bacteria can have the following aspects: one is to use natural luminescent bacteria to perform toxicity testing, the luminous intensity of bacteria is negatively correlated with the concentration of toxic substances (called light-off), and the comprehensive toxicity is reflected by the luminous inhibition rate , this method has been widely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/113C12N15/11C12N15/63C12R1/42
Inventor 张国霞张家强孙国萍许玫英蒋雍君郭诗韵
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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