Arsenite inhibitory factor reporter gene plasmids, construction method and application thereof

A technology of arsenite and inhibitors, which is applied in the fields of genetic engineering and molecular biology, can solve problems such as high background and achieve good inductive effects

Active Publication Date: 2018-05-22
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem with the β-galactosidase reporter gene is that its natural occurrence in bacteria can lead to high background

Method used

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  • Arsenite inhibitory factor reporter gene plasmids, construction method and application thereof
  • Arsenite inhibitory factor reporter gene plasmids, construction method and application thereof
  • Arsenite inhibitory factor reporter gene plasmids, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Plasmid construction

[0032] A series of high copy number plasmids derived from pGFPuv (Clontech) containing the pUC origin of replication (ColE1). The firefly luciferase gene was used to replace the GFPuv gene at the XbaI and EcoRI sites, and the lac promoter sequence was eliminated by digestion with HindIII and PvuII. A fragment containing the 91 bp promoter region of the R773ArsR operon was synthesized, and the luciferase gene upstream of HindIII and XbaI was cloned to obtain the pLHPars4 vector. A fragment containing a 91bp promoter region and a fragment encoding amino acids 1-102 of ArsR were synthesized and cloned to obtain pLHPars5. Fragments containing the ArsR binding sequence in Escherichia coli chromosome (EC-BS) or the binding sequence in acidic Thiobacillus ferrooxidans (AF-BS) were synthesized and inserted between the HindIII and PvuII sites of pLHPars5 , were cloned to obtain pLHPars7 and pLHPars10. In addition, fragments of EC-BS / AF-BS, two parts ...

experiment example 1

[0039] Experimental example 1 using high copy number plasmids to achieve high levels of luciferase gene expression

[0040] To assess the arsenite-inducibility of the luciferase gene, transformed cells were treated with 10 μM sodium arsenite for 2 hr; as figure 1 As shown, no significant arsenite-mediated induction of luciferase activity was observed. Escherichia coli chromosome ArsR can be used as a trans-regulator for combining with Escherichia coli chromosome and plasmid R773 operon. Thus, a simple explanation for the absence of induction is that the free ArsR predominates compared to the ArsR-bound promoter / operator sequence due to the high copy number plasmid in cells providing high levels of luciferase expression. Addition of arsenite, which removes ArsR from the promoter / operator, did not alter luciferase activity significantly compared to the unbound form, due to the very limited number of promoters / operons bound to ArsR .

[0041] Experimental Example 2 The co-expr...

experiment example 3

[0043] Experimental Example 3 Adding the ArsR binding sequence in front of the Ars operon

[0044] A common approach in mammalian systems is to genetically insert an additional copy of the cis-acting element in front of the promoter to better measure the inducibility of the reporter gene. This approach has been used to reduce the basal background of reporter gene activity during arsenite biosensor construction by inserting an extra copy of the E. coli ArsR binding sequence between the ArsR gene and the reporter gene. It showed slightly lower background compared to the control reporter without the addition of the additional ArsR binding sequence. In the present invention, an Escherichia coli ArsR binding sequence (EC-BS) or acidic Thiobacillus ferrooxidans ArsR binding sequence (AF-BS) is constructed in front of the R773ArsR promoter / operon of pLHPars5 to obtain reporter gene vectors pLHPars7 and pLHPars10( figure 1 ). Treatment of DH5α-transformed cells with 10 μM arsenite ...

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Abstract

Arsenite inhibitory factor reporter gene plasmids, construction method and application thereof are disclosed. The arsenite inhibitory factor reporter gene plasmids are pLHPars9 and pLLPars9, the nucleotide sequences of which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The arsenite inhibitory factor reporter gene plasmids of the invention are very sensitive to arsenite, and the detection range is 0.04-50 [mu]M, which exhibits a wide dynamic detection range. Therefore, the arsenite inhibitory factor reporter gene plasmids of the invention can be used as biosensors for the detectionof arsenite. Furthermore, the arsenite inhibitory factor reporter gene plasmid pLHPars9 of the invention can be used for the detection of antimonite in addition to arsenite.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and molecular biology. More specifically, the invention relates to a kind of arsenite inhibitor reporter gene plasmid and its construction method and application. Background technique [0002] As a naturally occurring element, arsenic is widely distributed throughout the environment in inorganic or organic forms, the inorganic form being highly toxic. The drinking water target set by the World Health Organization is 10 μg / L, but in some countries, groundwater is polluted by arsenic concentrations higher than the allowable value, which poses a threat to the health of citizens. Long-term exposure to arsenic from drinking water and food has been linked to a variety of diseases, including cancer, cardiovascular disease, neurotoxicity, and diabetes. To prevent further arsenic exposure, rapid, cost-effective in situ analytical techniques to monitor arsenic in water supplies are necessary. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N1/21C12Q1/6876C12R1/19
Inventor 李先强方云姜昕许玫英郭俊
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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