sacb-mediated genetic manipulation of Pseudomonas aeruginosa
A technology of Pseudomonas chlororaphis and genetic manipulation, which is applied in the field of genetic manipulation of biocontrol bacteria and can solve the problems of different genetic transformation conditions and the like
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Embodiment 1
[0049] Example 1 Sensitivity determination of Pseudomonas aeruginosa YL-1, Escherichia coli DH5α and S17-1 to ampicillin, gentamicin and tetracycline
[0050] The Pseudomonas aeruginosa YL-1 used in this example is a strain isolated and obtained by the applicant to a variety of important pathogenic bacteria in agriculture (Burkholderia glumae; Erwinia amylovora; Erwinia amylovora; Pectobacterium carotovora, etc.) and pathogenic fungi (Rhizoctoniasolani, etc.) have strong inhibitory effects on biocontrol bacteria (Liu Youzhou et al., Journal of Phytopathology, 2015).
[0051] Pseudomonas aeruginosa YL-1 was cultured in LB liquid medium at 28°C and 180rpm on a shaker overnight, and the concentration was about 10 8 cfu / mL bacterial solution for use. After gradient dilution, the bacterial solution was spread on LB plates with ampicillin concentrations of 10, 50, and 100 μg / mL, and LB plates with gentamicin and tetracycline concentrations of 10, 25, and 50 μg / mL, respectively, and...
Embodiment 2
[0056] Example 2 Construction and Connection of Target Gene Deletion Fragments
[0057] In order to mine the active substances of Pseudomonas aeruginosa YL-1 antagonizing pathogenic bacteria and fungi and locate its synthetic gene cluster, in this example, the synthetic gene of fluorescent siderophil (Pyoverdine) in Pseudomonas aeruginosa YL-1 was used. The pvdS gene on the cluster is the target gene, which is a sigma factor and regulates the transcription of siderophil in strain YL-1. Once the pvdS gene is destroyed, strain YL-1 cannot synthesize siderophil. The green color characteristic of the wild-type strain cannot be displayed.
[0058] Table 2 Primer table used in Example 2
[0059]
[0060] (1) Select the flanking sequences at both ends of the target gene pvdS, use Bioxm software to design upstream and downstream primers F1, R1, F2 and R2, respectively, add HindIII, KpnI, XbaI restriction sites, as shown in Table 2; (2 ) CTAB method ("Molecular Cloning Experiment ...
Embodiment 3
[0088] Example 3 Genetic transformation of ligation product carrying fusion gene into Escherichia coli DH5α
[0089] (1) Preparation of Escherichia coli DH5α chemically competent cells
[0090] ①Pick a fresh single colony of Escherichia coli in 25mL LB liquid medium, cultivate overnight at 37°C and 220rpm;
[0091] ② Transfer the cultured bacterial liquid to 50mL of fresh LB liquid medium at 1:100, 37℃, 220rpm, 3-3.5h to OD 600nm ≈1.0;
[0092] ③ After ice bathing the above bacterial liquid for 10 min, centrifuge at 4°C and 6000 rpm for 10 min, discard the supernatant, and leave the precipitated bacterial cells;
[0093] ④Add pre-cooled CaCl 2 (0.1M) 10mL, gently blow to suspend the bacteria, ice bath for 30min;
[0094] ⑤ Centrifuge at 6000 rpm for 10 min at 4°C, discard the supernatant, and leave the precipitated bacteria;
[0095] ⑥Add pre-cooled CaCl 2 (0.1M containing 15% glycerol) 1mL, gently pipetting to suspend the cells. Divide into 1.5mL centrifuge tubes, abou...
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