Construction method of microbial sensor and application method of microbial sensor
A technology of a microbial sensor and a construction method, applied in the field of microbial sensor construction, can solve problems such as quantitative detection that have not yet been seen, and achieve the effects of reducing detection difficulty, high sensitivity, and improving sensitivity
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Embodiment 1
[0044] figure 1 It is the schematic diagram of the preparation circuit of the microbial sensor in Example 1 of the present invention, such as figure 1 Shown, the invention provides a kind of construction method of microbial sensor, comprises the following steps:
[0045] Step 1 constructing a deficient strain of the amino acid to be tested;
[0046] Step 2 Construct a plasmid with a fluorescent protein gene as a reporter gene, intercept the target gene fragment required for the LuxI / LuxR cyclic amplification circuit, connect and construct, obtain a plasmid containing a cyclic amplification circuit based on the LuxI / LuxR system, and transform the recombinant plasmid into a competent state In the bacterial strain obtained in step 1, the reporter gene fragment is repeatedly expressed to obtain a microbial sensor with signal cycle amplification capability;
[0047] Step 3: Cultivate the strain obtained in Step 2 to the logarithmic growth phase, and perform starvation culture in ...
Embodiment 2
[0058] The preparation method of the microbial sensor with the function of circularly amplifying the signal takes lysine as the amino acid to be detected and GFP as the reporter gene.
[0059] Step 1: Construction of lysine-deficient Escherichia coli MG1655
[0060] (1) Select the 50 bp sequences on both sides of the lysA gene to be knocked out as the upstream and downstream homologous recombination arms of gene targeting. The upper and lower homologous recombination arms are designed to be located on both sides of the specific primers for the kanamycin resistance gene (kan), and long primers that can be used for PCR amplification are obtained by chemical synthesis.
[0061] (2) Using the plasmid pKD4 as a template, the lysA gene targeting fragment was obtained by high-fidelity PCR amplification with the above-mentioned long primers.
[0062] (3) Preparation of electroporation competent cells of MG1655 strain. The pKD46 helper plasmid of the Lambda Red recombination system w...
Embodiment 3
[0092] In the present invention, the amino acid to be tested is a monomeric amino acid or a polypeptide. Taking lysine as an example, a lysine polypeptide is used because the polypeptide can be hydrolyzed into multiple lysine molecules, and it is easier to make lysine Acid-deficient E. coli grows, making it easier to scale up the assay. The polypeptide sequences used are CKKKKK for CK5, CKKKKKKKKKK for CK10, and CKKKKKKKKKKKKKKK for CK15.
[0093] The microbial sensor is starvation-treated lysine-deficient Escherichia coli with gfp as the reporter gene, and the original lysine molecules have been depleted. At this time, different groups of lysine polypeptide molecules with different concentrations are added to the M9 medium. The amplification response of lysine polypeptide molecules relative to lysine can be determined, and the specific steps are as follows:
[0094] (1) Prepare three kinds of lysine polypeptide solutions of CK5, CK10 and CK15 with deionized water, filter and...
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