(-)-[gamma]-lactamase, gene, mutant, vector as well as preparation method and application of (-)-[gamma]-lactamase
A technology of lactamase and lactam, applied in the field of bioengineering, can solve problems such as difficult large-scale application, low product concentration, and large catalyst usage
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Embodiment 1
[0045] Example 1 Cloning of (-)-γ-lactamase SvGL gene
[0046] The strain Streptomyces viridochromogenes CGMCC 4.692 was cultured in LB medium, and the high-purity and large-fragment genomic total DNA was extracted by the high-salt method, dissolved in TE buffer (pH8.0), and stored at -20°C. The specific method Refer to the "Refined Molecular Biology Experiment Guide" edited by F. Osper et al.
[0047] The extracted total DNA was partially cleaved with restricted enzyme Sau3AI, and the cleaved DNA fragments were purified by electrophoresis, and about 4-6kb fragments were recovered using a gel recovery and purification kit, and the recovered DNA was dissolved in TE buffer (10mM ,pH8.0), stored at -20°C.
[0048] Connect with the vector pUC118 according to the following reaction system:
[0049]
[0050]
[0051] Incubate at 16°C for 6 hours, take 10 μL of the enzyme-linked product and transform 200 μL of Escherichia coli DH5α competent cells (TaKaRa, Code: D9057), pick ...
Embodiment 2188
[0053] Example 2 Site-directed saturation mutation of 188-position phenylalanine
[0054] use II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) described the protocol to operate. Primers containing mutation points were designed: forward primer (Primer F) 5'-GCCGTCCGCAACAGCNKAACGTCGCCGCGGGC-3' and reverse primer (Primer R) 5'-GCCCGCGGCGACGTTMNNGCTGTTGCGGACGGC-3'. PCR reaction system (50μL): template 0.5~20ng, 5μL 10×KOD plus buffer, 5μL dNTP (each 2.0mM), 2μL MgSO 4 (25mM), 1 μL (20 μM) of each pair of mutant primers, 1 unit of KOD enzyme (TOYOBO CO., LTD., Osaka, Japan), add sterilized distilled water to 50 μL. The template described therein is the plasmid pET28a-SvGL obtained in Example 1. PCR reaction program: (1) denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 sec, (3) annealing at 55°C for 1 min, (4) extension at 68°C for 7 min, steps (2) to (4) were performed for 30 cycles in total, Finally, extend at 68°C for 10 min, and store the produc...
Embodiment 3
[0055] Example 3 Site-directed saturation mutation of leucine at position 130
[0056] use II Site-Directed Mutagenesis Kit (Stratagene, Catalog #200522) described the protocol to operate. Primers containing mutation points were designed: forward primer (Primer F) 5'-TCGCTGGAGCCCTGNNKCTCAAGTCCGACGAC-3' and reverse primer (Primer R) 5'-GTCGTCGGACTTGAGMNNGCAGGGCTCCAGCGA-3'. PCR reaction system (50μL): template 0.5~20ng, 5μL 10×KOD plus buffer, 5μL dNTP (each 2.0mM), 2μL MgSO 4 (25mM), 1 μL (20 μM) of each pair of mutant primers, 1 unit of KOD enzyme (TOYOBO CO., LTD., Osaka, Japan), add sterilized distilled water to 50 μL. Wherein said template is the plasmid pET28a-SvGL that embodiment 2 obtains F188W . PCR reaction program: (1) denaturation at 94°C for 5 min; (2) denaturation at 94°C for 30 sec, (3) annealing at 55°C for 1 min, (4) extension at 68°C for 7 min, steps (2) to (4) were performed for 30 cycles in total, Finally, extend at 68°C for 10 min, and store the produc...
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