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A kind of recombinant bacteria and its application

A technology of recombinant bacteria and Arthrobacter, applied in the field of genetic engineering, can solve the problem of inhibitors with many manpower and material resources, and achieve the effect of good promotion and use value, high efficiency and simple operation

Inactive Publication Date: 2016-09-07
COLD & ARID REGIONS ENVIRONMENTAL & ENG RES INST CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The screening of many inhibitors took a lot of manpower and material resources

Method used

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  • A kind of recombinant bacteria and its application
  • A kind of recombinant bacteria and its application
  • A kind of recombinant bacteria and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of a recombinant plasmid containing the mTFP-ftsZ gene labeled with cyan fluorescence

[0033] 1. Cloning of mTFP gene

[0034] The mTFP cyan fluorescent protein gene was found from the NCBI gene bank. The designed upstream and downstream genes (upstream primer P1aaggatccgATGGTCAGCAAGGGCGAAGAAACG, downstream primer P2tttctagaCTACTTCAGGAAGTCGGGGACATCC) were synthesized by Beijing Huada Gene Technology Co., Ltd. and amplified by conventional PCR method. PCR reaction system 25 μL : Proof DNA polymerase 2μL, 1×Proof buffer 2.5μL, 25 mmol / L MgCl 2 3μL, 10 mmol / L dNTP 0.5μL, DNA template 2μL, each primer 10pmol, the rest is water. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, extension at 68°C for 30 s, and 30 cycles of reaction; after the reaction, the target fragment of the 708b mTFP gene was obtained.

[0035] 2. Cloning of ftsZ gene

[0036] Extract the total DNA from Arthrobacter sp.A3, use i...

Embodiment 2

[0042] Example 2 Verification of the two-color fluorescence system

[0043] 1. Transfer pART2-mTFP-ftsZ into Arthrobacter sp.A3

[0044] Transfer pART2-mTFP-ftsZ into Arthrobacter sp.A3 competent cells by electric shock, electric shock conditions: 2500V / cm, 25μF, 700Ω, 2mm electric shock cup, 200μl competent cells, 1μl recombinant plasmid, after electric shock, use LB liquid medium +0.5 M sorbitol 800μl was incubated for 6-8h. Pick out the grown single colony and shake the bacteria, and carry out colony PCR verification, the primer used is 5P ftsZ 5'- GTGGCAGCACCCCAGAATTACTTGGC-3' and 3P ftsZ 5'-CTACTTCAGGAAGTCGGGGACATCCA-3'. PCR reaction system 25 μL: Proof DNA polymerase 2 μL, 1×Proof buffer 2.5 μL, 25 mmol / LMgCl2 3 μL, 10 mmol / L dNTP 0.5 μL, DNA template 2 μL, each primer 10 pmol, the rest is water. The PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 sec, annealing at 55°C for 30 sec, extension at 68°C for 60 sec, and 30 cy...

Embodiment 3

[0049] Example 3 Inhibition of ftsZ-related rapid detection test for antibiotics

[0050] Arthrobacter sp.A3 / pART2-mTFP-ftsZ was inoculated with LB liquid medium containing kanamycin at a final concentration of 140 μg / ml (the formulation of LB liquid medium was: tryptone 1%, yeast extract 0.5%, chlorine Sodium chloride 1%, 140 μg / ml kanamycin, the rest is distilled water, adjusted to pH 7.0, sterilized at 121°C for 20 min), 200r / min, 20°C shaker culture to the early and mid-logarithmic growth phase, and the The test compound PC190723 and DMSO were added to the above-mentioned LB liquid medium respectively, (if multiple compounds are to be detected simultaneously, each compound needs to be mixed and then added), 1 mL was centrifuged, the precipitate was washed twice with PBS, and then washed with 20 μL of the mixed solution ( The mixed solution was 1 / 100 (V / V) vancomycin and the final concentration was 1 μg / ml vancomycin-fluorescent and 1 / 100 (V / V) DAPI (4',6-diamidino-2-pheny...

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Abstract

The invention provides a recombinant bacterium containing mTFP‑ftsZ recombinant Arthrobacter Arthrobacter sp .A3, mTFP‑ftsZ The gene sequence of the gene is shown as SEQ ID No.1 in the sequence listing. The present invention also provides a method for rapidly detecting and inhibiting antibiotics related to FtsZ, which is to insert the compound to be tested into Arthrobacter, cultivate to the early and middle logarithmic growth phase, and then fluorescently label the new cell wall, compare it with a blank control, and observe The location and ratio of the new cell wall ring, if the formation or ratio of the new cell wall ring changes, the compound to be tested has the ability to inhibit FtsZ-related antibiotics. The method provided by the invention can detect whether there is an ftsZ-inhibiting active substance in a fermentation product or a variety of chemical derivatives at a time, and has the advantages of simple operation and high efficiency, and a definite result can be obtained in only 2 hours, and has good popularization and use value.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant bacterium and its application. Background technique [0002] Since the first discovery of antibiotics - penicillin in 1928, in the past few decades, the antibiotic industry has developed rapidly and made great contributions to human health. However, due to the irrational use of antibiotics, especially in my country, the problem of antibiotic resistance has gradually broken out. To solve this problem, in addition to paying attention to the rational use of antibiotics, it is also very important to develop new antibiotics that have inhibitory activity against drug-resistant bacteria. [0003] At present, there are two main methods to study new antibiotics. One is to carry out comprehensive structural modification of existing antibacterial drugs. They act on bacterial target sites, including bacterial protein synthesis, nucleic acid synthesis, cell wall s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C07K14/195G01N21/64C12R1/06
Inventor 陈熙明张昺林张威张满效陈拓刘光琇
Owner COLD & ARID REGIONS ENVIRONMENTAL & ENG RES INST CHINESE
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