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
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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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