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Fluorescent strain E. coli C600 and construction method and application thereof

A construction method and fluorescence technology, applied in the field of genetic engineering, can solve the problems of inability to use, inability to utilize lactose, and require a lot of work, and achieve the effect of strong stability

Active Publication Date: 2019-07-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, another part of Escherichia coli and all Salmonella and other bacteria still grow white colonies on MacConkey agar because they cannot utilize lactose, which brings certain difficulties to the screening of E.coli C600 (zygotes) obtained from foreign plasmids , and the existing main method for identifying zygote and donor bacteria is to identify zygote and donor bacteria through bacterial genome repeat sequence PCR (ERIC PCR)
ERIC PCR is time-consuming and labor-intensive
If the electrophoresis patterns of the ERIC PCR products of the donor bacteria and the zygote are consistent, the method of ERIC PCR cannot be effectively determined as a conjugation
In addition, some recipient bacteria cannot grow on MacConkey agar medium, and MacConkey selection medium cannot be used to screen zygotes, which seriously affects the accuracy and accuracy of zygote screening and increases the workload of scientific research

Method used

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  • Fluorescent strain E. coli C600 and construction method and application thereof
  • Fluorescent strain E. coli C600 and construction method and application thereof
  • Fluorescent strain E. coli C600 and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The construction of embodiment 1 fluorescent E.coli C600-1

[0057] The method for constructing E.coli C600-1 based on ISApl1 insertion sequence transposition mutation technology, specifically as follows figure 1 shown.

[0058] 1. Construction of pTFX-RP4-lux plasmid

[0059] The present invention inserts the suicide type R6k replicon (GenBank accession number: MH626522.1.(504...892)), Lux gene cluster (GenBank accession number: KX670548.1.(3429...9187)), ISApl1 insertion sequence (GenBank accession number: MH924589.1.(219,280..220,203)), tellurite resistance gene (tpm) (GenBank accession number: KX397287.1.(4,473..5,126)), RP4 conjugative transfer gene (GenBank Accession number: AJ868289.1.(147..1,876)) were combined to construct a new plasmid pTFX-RP4-lux.

[0060] The following is one of the possible construction methods, but not limited to it:

[0061] Such as figure 2 Shown, the construction process of the pTFX-RP4-lux plasmid that the present invention comp...

Embodiment 2

[0086] Morphological observation of embodiment 2 fluorescent bacterial strain E.coli C600-1

[0087] The E.coli C600-1 that embodiment 1 obtains at tpm R After the resistant LB agar plate was cultured for 20 hours, at tpm R The morphology grown on the resistant LB agar plate is as follows Figure 4 As shown, black colonies of irregular size grew.

[0088] the tpm R The formula of the resistant LB agar plate is tryptone 10.0g / L, yeast extract powder 5.0g / L, sodium chloride 10.0g / L, agar 15.0g / L, pH value 7.0±0.2, final concentration 25μg / mL tellurite Sodium acid.

[0089] Will Figure 4 The agar plate is passed through a small animal living imager, and bacteria can be seen to produce biological fluorescence, such as Figure 5 .

[0090] The E.coli C600-1 that embodiment 1 obtains is on LB agar plate (prescription is tryptone 10.0g / L, yeast extract powder 5.0g / L, sodium chloride 10.0g / L, agar 15.0g / L, pH value 7.0±0.2), the colony was beige.

[0091] The E.coli C600-1 t...

Embodiment 3

[0092] Example 3 Verifies the ability of E.coli C600-1 to obtain exogenous DNA

[0093] The E.coli C600-1 (recipient bacterium) obtained in Example 1 and 12 clinical plasmid-mediated MCR-1 colistin-resistant Escherichia coli (donor bacteria, see Table 1 for details), the donor Somatic bacteria have been published in the literature: Sun J, Fang L X, Wu Z, et al. Genetic Analysis of the IncX4Plasmids: Implications for a Unique Pattern in the mcr-1 Acquisition[J]. Scientific Reports, 2017, 7(1): 424.

[0094] Table 1 Statistical table of bacterial markers

[0095]

[0096] Receive single colonies into 4mL LB test tube broth, shake culture at 37°C until logarithmic phase of growth, mix donor bacteria and recipient bacteria at a volume ratio of 1:1, take 20 μL and drop them into 24-well plates (perfusion per well 900μL LB agar), after static culture at 37°C for 4 hours, dilute the mixed cultured hatch with physiological saline to a suitable gradient (the number of colonies on t...

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Abstract

The invention provides a fluorescent strain E. coli C600 and a construction method and application thereof. By constructing a recombinant plasmid containing an R6k replicon, a transposition unit and an RP4 conjugative transfer gene, the recombinant plasmid is transferred to a host bacterium in which the recombinant plasmid can replicate to obtain a recombinant strain, the recombinant strain and E.coli C600 are subjected to mixed culture, and then the fluorescent strain E. coli C600 can be obtained through screening, wherein the transposition unit is formed by using two ISApl1 for sandwichinga Lux gene cluster and a tellurite drug-resistance gene in the middle. The exogenous gene acquiring capability of the fluorescent strain E. coli C600 is not influenced, the stability is high, stable inheritance can be ensured, the fluorescent property cannot lose along with bacterium passage, and thus the fluorescent strain E. coli C600 can serve as a recipient bacterium for conjugation experiments. By adopting the fluorescent strain E. coli C600, recipient bacteria and zygotes can be identified more efficiently and more intuitively in the fields related to plasmid conjugational transfer, andtherefore the fluorescent strain E. coli C600 has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to fluorescent labeling technology, in particular to a fluorescent bacterial strain E.coli C600 and its construction method and application. Background technique [0002] A plasmid is a small circular double-stranded DNA molecule that can replicate independently. It can spread not only between bacteria, but also between other organisms, such as archaea and fungi. Genes carried on plasmids often confer specific capabilities on the host organism, such as: antibiotic resistance, heavy metal resistance or pathogenic factors. Plasmids can often be transmitted naturally to other bacteria. Transmission is possible even between different species, which allows them to spread easily and at high speed. It is a commonly used method in molecular biology to study the zygosity and mobility of clinical plasmids through conjugation experiments, and then to study the transmission mechanis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
CPCC12N9/0071C12N15/52C12N15/70C12N2800/90C12Y114/14003
Inventor 孙坚李龚何玉张苗媛媛杨心怡廖晓萍刘雅红
Owner SOUTH CHINA AGRI UNIV
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