Polycyclic aromatic hydrocarbon degradation gene engineering strain, and construction method and application thereof
A technology of genetically engineered strains and genetically engineered bacteria, applied in the field of polycyclic aromatic hydrocarbons degrading genetically engineered strains, can solve problems such as inability to guarantee lethality, and achieve the effects of reducing biosafety risks and improving controllability
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[0044] Example 1: Fusion of lac promoter and nuclease gene nuc
[0045] Amplify the nuc fragment with primers nuc-f and nuc-r; PCR reaction system (50μl): template DNA 1.0μl; HS Premix (Baoshenggong, Dalian) 25.0μl; 50μM primer 0.5μl; ddH 2 O 23.0μl; PCR reaction program: 94°C pre-denaturation 3min; (94°C pre-denaturation 30s—57°C annealing 5s—72°C extension 90s)×30 cycles; 72°C extension 10min. See the amplification results figure 1 ( figure 1 : PCR amplification of nuc gene, where M: 5000bp DNA Marker, 1: nuc gene fragment). Gel electrophoresis imaging showed specific amplified bands, and the expected fragment length (801bp) was consistent. Use Nde I and HindIII to digest the amplified products of the vector pUC-18 and nuc fragments. After ligation, the plasmid pUC-nuc is transformed into E.coli DH5α. After the plasmid is amplified and extracted, the nuc fragment can be amplified using it as a template. Use Nde I and Hind III enzyme digestion to see pUC-18 vector and nuc frag...
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[0046] Example 2: Construction of recombinant transposon vector pUT-lacnuc
[0047] The lacnuc gene and the vector pUT / mini-Tn5Km were digested with Not I restriction enzyme, the lacnuc fragment was recovered after purification and ligated with pUT / mini-Tn5Km, and the ligation product was transformed into E.coli DH5αλpir. Pick the positive clones on the plate and use the primers lacnuc-f and lacnuc-r to perform colony PCR for verification, and the verification results are positive (see Figure 4 : PCR verification of pUT-lacnuc vector, where M: 10000bp DNA Marker, 1: lacnuc gene fragment); At the same time, the extracted plasmid was digested with Not I, and the gel electrophoresis results showed that there were two near 7.1kb and 1kb Strip (see Figure 5 : Restriction digestion identification of pUT-lacnuc vector, where M: 5000bp DNA Marker, 1: Not I digestion product), corresponding to pUT / mini-Tn5Km plasmid and lacnuc gene, respectively, proving the successful construction of pU...
Example Embodiment
[0048] Example 3: Construction of genetically engineered strain Pseudomonas putida GLEB3
[0049] The fusion gene lacnuc was recombined into the Pseudomonas putida GLB3 chromosome by means of three-parent binding. The strains required for the operation are as follows: acceptor bacteria (Pseudomonas putida GLB3), helper bacteria (E.coli DH5α (pRK2073)), donor bacteria (E. .coli DH5α(pUT-lacnuc)). The mixing ratio of each strain is 1:2:1, and the mixing system uses 10mM MgSO 4 Solution, the plate medium used should be LB plate medium containing kanamycin (25mg / L) and chloramphenicol (25mg / L). After culturing until the colonies are visible, pick the colonies and use the primers lacnuc-f and lacnuc-r to verify the colony PCR of the lacnuc gene. Image 6 It shows that the lacnuc gene has been successfully transformed into the recipient strain Pseudomonas putida GLB3, and the recombinant strain was named Pseudomonas putida GLEB3 (preservation number CGMCC No. 13014). Propagate the rec...
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