Preparation method of biosensor with high sensitivity to heavy metal copper and product obtained thereby

Abstract

The invention relates to a preparation method of an Escherichia coli (E.coli) biosensor and a product obtained thereby. The preparation method comprises the following steps: (1) knocking out three genes, namely copA, cueO and cusA in a wild-type E.coli MC4100 genome by a Red recombination system; (2) constructing a fusion reporter gene PcopA::gfpmut2 by adopting a cross PCR (Polymerase Chain Reaction) technology; (3) cloning the segment obtained in step (2) to a pMD18-T vector; (4) carrying out double digestion on a PcopA::gfpmut2-T vector to obtain a target strip, and then connecting the target strip to a pET28a vector; and (5) converting a fusion reporter vector obtained in the step (4) to a deltacopA-deltacueO-deltacusA tri-gene mutant strain obtained in the step (1). In the preparation method, the three genes such as the copA, the cueO and the cusA are deleted from wild-type E.coli MC4100, and then the genes are converted to the fusion reporter vector PcopA::gfpmut2-pET28a so as to finally obtain the product. The product obtained by the method has the characteristics of high specificity, high sensitivity, high speed, high efficiency, high cost performance and the like, and is simple and convenient to use, thus being applicable to real-time field monitoring of copper ions.

Description

technical field [0001] The invention relates to a bacterial strain transformed by metabolic engineering by means of molecular biology, in particular to a preparation method of an Escherichia coli biosensor highly sensitive to heavy metal copper ions and a product thereof. Background technique [0002] Copper is an essential trace element for animals and plants. Copper deficiency in the human body can cause anemia, diarrhea and other diseases, but excessive copper can also cause harm to humans, animals and plants. Studies have found that excessive heavy metal copper is toxic to all living organisms. Excessive deposition of copper in the human body is involved in the occurrence and development of many diseases. Copper can accumulate in vital organs of the human body, such as liver, kidney, brain, etc., especially in certain Some people with chromosomal recessive diseases, due to the dysfunction of bile excretion of copper, copper accumulates in the liver, causing liver damage,...

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to construct a highly sensitive Escherichia coli biosensor for copper ions through gene knockout and gene fusion technology to quickly and sensitively detect copper ions, so as to solve the sensitivity of biological detection methods using wild-type Escherichia coli as a host bacterium It is not a high-level problem, and can overcome the disadvantages of physical and chemical methods such as cumbersome operations, special instruments and laboratory equipment.

Images