Biocontrol lysobacter enzymogenes OH11 sourced quorum sensing quenching protein and application thereof
A quorum sensing and protein technology, applied in the field of microorganisms, can solve problems such as limited use, high cost of use, and poor heat resistance
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Embodiment 1
[0028] The construction of embodiment 1 recombinant expression vector
[0029] 1) Construction of pBBR-Le0959 expression vector
[0030] Using the genomic DNA of the OH11 strain as a template, the target gene Le0959 was amplified by PCR using primers pBBR-Le0959-F and pBBR-Le0959-R (Table 1), and the fragment was double-digested with restriction endonucleases HindIII and XbaI. T4 ligase was ligated to the pBBRMCS-5 vector that was also cut with HindIII and XbaI, and the ligated product was transformed into Escherichia coli DH5α strain after heat shock, spread on LB solid plates containing Gm 25 μg / mL, and cultured at 37°C for 24 After 1 hour, select the grown transformant, and carry out PCR verification with primers pBBR-F and pBBR-R, and the electrophoresis is as follows: figure 1 shown. The transformant with the correct band size was sent to Suzhou Jinweizhi Company for sequencing. After the sequencing was correct, the plasmid was extracted to obtain the recombinant plasmi...
Embodiment 2
[0034] The acquisition of embodiment 2 recombinant bacteria and control bacteria
[0035] 1) Preparation of electroporation competent
[0036] Pick a single colony of Lysobacterium zymolyticum OH11 from a solid petri dish and inoculate it in LB liquid medium for overnight culture at 28°C, transfer it to fresh 50mL LB liquid medium at a ratio of 1:50 and continue to cultivate until the OD600 is 0.6- 0.8; place on ice for 1 hour and then centrifuge at 6000rpm at 4°C to collect the cells; suspend the cells with pre-cooled sterilized 10% glycerin solution, centrifuge at 6000rpm at 4°C to collect the cells; repeat the previous step twice ;Suspend the bacteria with 2mL of 10% glycerol solution and divide into 2mL sterilized centrifuge tubes, 100μL per tube, freeze them with liquid nitrogen and store them at -80°C for later use. Fluorescent pseudomonas 2P24 wild type and ΔpcoI mutant were prepared by the same method to prepare competent cells.
[0037] 2) Acquisition of recombinant...
Embodiment 3
[0041] Example 3 Detection of AHL Signaling Molecules
[0042] 1) 2P24 recombinant bacteria pBBR-Le0959 and pBBR-gfp growth curve detection
[0043] Pick a single colony of 2P24 recombinant pBBR-Le0959 and pBBR-gfp from a solid petri dish, inoculate them in LB medium for overnight shaking culture, transfer to fresh LB liquid medium at a ratio of 1:50 and continue to grow to OD 600 1; then transferred to fresh 50mL LB liquid medium at a ratio of 1:100 for culture, each bacterium was inoculated into 3 bottles as a parallel experiment, and 200 μl was sampled every 2 hours to detect the absorbance value of each strain at OD of 600nm. The result is as figure 2 As shown, the growth rate of 2P24 recombinant pBBR-Le0959 and pBBR-gfp had no significant difference; therefore, the experiment showed that the expression of recombinant plasmid pBBR-Le0959 had no significant effect on the growth of Pseudomonas fluorescens 2P24.
[0044] 2) Detection of AHL signaling molecules in 2P24 reco...
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