Separated 5-enolpyruvyl shikimate-3-phosphate synthase gene
A technology of enolacetone shikiki and phosphate synthase, applied in the field of genetic engineering
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Embodiment 15
[0032] Example 15-Synthesis and Cloning of Enolpyruvyl Shikimate-3-Phosphate Synthase Gene
[0033] (1) AroA I.vari Synthesis of Gene Sequence
[0034] AroA encoding EPSPS of Isoptericola Variabilis strain obtained in Genbank database I.vari Gene sequence, the nucleotide sequence 1374bp of the gene was synthesized by Nanjing GenScript and provided with recombinant plasmid pUC57-AroAI.vari ( Figure 4 ). Its nucleotide sequence and encoded amino acid sequence are shown in the sequence listing SEQ ID NO:1 and SEQ ID NO:2.
[0035] (2) AroA I.vari Cloning of Gene Sequences
[0036] According to AroA I.vari Sequence design primers: add a BamHI restriction site at the 5' end of the primer, add a restriction site EcoRI at the 3' end, and the DNA sequence of the primer pair is as follows:
[0037] Forward primer (5'-AroA I.vari -BamHI):5'-CGC GGATCC ATGACGCCAGCGCCCGCCAGC-3', where the underlined part is the restriction site;
[0038] Reverse primer (3'-AroA I.vari -EcoRI)...
Embodiment 2
[0053] Expression, purification and analysis of embodiment 2EPSPS protein
[0054] (1) Expression of the target protein EPSPS
[0055] The recombinant plasmid pGEX-6p-1-AroA I.vari Transformed into expression host cell E. coli BL21 (DE3). The identified positive clones were activated overnight, transferred to 1L LB liquid medium containing 100μg / mL Ampicillin at an inoculation volume of 1% by volume, and cultured at 37°C for 2-3h until OD 600 When it reaches about 0.6, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.5mM, and cultured at 22°C and 180rpm for 25h. Collect the bacteria by centrifugation, then adjust the pH to 7.0 with Hepes buffer (recipe: 50mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), 2mM dithiothreitol (DTT), make up double-distilled water to 1L, set aside ) to wash the cells once, suspend them with 50 mL of Hepes buffer, and break the cells with a high-pressure cell disruptor (purchased from GEA Niro S...
Embodiment 3
[0075] Example 3: Genetic transformation experiment (functional complementation verification)
[0076] The recombinant plasmid pGEX-6p-1-AroA J.limo Transformed into AroA gene-deficient Escherichia coli competent cells (for the transformation process, refer to the transformation process in the above-mentioned Example 1. This bacterial strain does not contain the AroA gene, which can eliminate the interference of the AroA gene contained in general E. coli) (Vaithanomsat and Brown2007), Transfer to M9 liquid medium containing 0mM, 50mM, 100mM, 150mM glyphosate (recipe: Na2HPO46.8g / L, KH2PO43.0g / L, NaCl0.5g / L, NH4Cl1.0g / L, MgSO4 7H2O0 .4929g / L, CaCl20.111g / L, glucose 4.0g / L, supplemented with double distilled water to 1L, adjusted the pH to 7.4 before sterilization, and sterilized under high pressure steam at 115°C for 10min) to measure the growth curve.
[0077] The constructed recombinant plasmid pGEX-6p-1-AroA I.vari Transform into competent cells Escherichia coli AroA-defic...
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