A genetic engineering strain producing p-hydroxycinnamic acid and its construction method and application
A technology of genetically engineered strains and hydroxycinnamic acid, applied in genetic engineering, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of high energy consumption, low product yield, low yield, etc., and achieve high production efficiency, The effect of simple conditions and easy operation
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Embodiment 1
[0045] Embodiment 1, the acquisition of phenylalanine deaminase expression construct
[0046] (1) Synthesize two primers respectively having the nucleotide sequences of SEQ ID No.: 5 and SEQ ID No.: 6 in the sequence table, and carry out PCR amplification with the cDNA of Arabidopsis thaliana as a template, and obtain the primers having the sequence encoding SEQ ID No.: DNA fragment of the nucleotide sequence of the sequence shown in 1. DNA polymerase was selected from Nanjing Nuoweizan Biotechnology Co., Ltd. Super-Fidelity DNA polymerase. The PCR amplification program was: 95°C for 5min; 94°C for 45s, 56°C for 45s, 72°C for 3.5min, a total of 30 cycles; 72°C for 10min, then drop to 10°C.
[0047] (2) PCR products were detected and separated by agarose gel electrophoresis. Under the irradiation of ultraviolet light, cut out the target DNA band. Then adopt the multifunctional DNA purification kit (spin column type) (Beijing Baitaike Biotechnology Co., Ltd.) to reclaim and...
Embodiment 2
[0049] Example 2, the acquisition of trans-cinnamic acid-4-hydroxylase expression construct
[0050] (1) Synthesize two primers respectively having the nucleotide sequences of SEQ ID No.:7 and SEQ ID No.:8 in the sequence listing. Two restriction sites NdeI and XhoI and their protective base sequences were respectively set at the 5'-ends of the synthesized primers SEQ ID No.:7 and SEQ ID No.:8, and PCR was carried out using the cDNA of Arabidopsis thaliana as a template Amplify. The PCR amplification procedure is the same as in Example 1. The PCR amplified products were detected by agarose gel electrophoresis, separated, gel-cut and recovered, and then digested with NdeI and XhoI, and ligated with T4 DNA ligase of Takara Bioengineering (Dalian) Co., Ltd. Digested pET29a vector (Novagen). The ligation product was transformed into Escherichia coli (E.coli) DH5α (purchased from Nanjing Novizan Biotechnology Co., Ltd.) competent cells, and spread on LB plates supplemented with ...
Embodiment 3
[0054] Embodiment 3, the acquisition of the expression construct that down-regulates the expression of pyridine nucleotide transhydrogenase SthA
[0055](1) Synthesizing two primers respectively having the nucleotide sequences of SEQ ID No.: 15 and SEQ ID No.: 16 in the sequence listing. PCR amplification was performed using the E. coli genome as a template to obtain a DNA fragment comprising the polynucleotide sequence shown in SEQ ID No.:4. The PCR amplification procedure is the same as in Example 1. PCR products were separated and recovered by agarose gel electrophoresis. Both ends of the recovered DNA fragments have 25 bp homologous sequences to both sides of the EcoRI restriction site of the pCL1920 vector.
[0056] (2) The pCL1920 vector was linearized with EcoRI restriction endonuclease (purchased from Bao Biological Engineering (Dalian) Co., Ltd. (TaKaRa)). Using the One-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), the obtained DNA frag...
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