Recombinant streptomyces diastatochromogenes with reinforced adpA expression, construction method and application
A technology for producing Streptomyces chromogenes and amylase, which is applied in the field of genetic engineering, can solve complex problems and other problems, and achieves the effect of enhancing the transcription level and improving the yield of toyomycin
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Embodiment 1
[0026] Example 1: Amplification of Target Gene and Construction of Recombinant Amylase Streptomyces Chromogenes
[0027] by S. diastatochromogenes 1628 chromosome genome as template, with PadpA F Nde I and PadpA R not I is a primer, PCR amplification obtains containing Nde I and not I two restriction sites adp A gene, connected with pMD18-T Vector to construct the cloning vector pMD18-T- adp A( Nde I + not I), the cloning vector pMD18-T- adp A( Nde I + not I) Transformed into recipient Escherichia coli, smeared on LB agar plate containing Amp, cultured overnight at 37 ℃, randomly picked positive transformants and sent them to Shanghai Sangong for sequencing and sequence analysis. Nde I and not I double digestion to obtain containing Nde I and not I two restriction sites adp A gene, connected with the same double-digested Streptomyces integrated shuttle expression vector pIB139 ( figure 1 ), the recombinant shuttle expression vector pIB1...
Embodiment 2
[0033] Example 2: The expression of AdpA gene plays a key role in the synthesis of toyocamycin toy Effect of F transcript levels
[0034] Recombinant amylase Streptomyces chromogenes 1628-ADPA and the original strain were cultured in CP medium for 48 hours, and the cells were harvested, and total RNA was extracted and DNA was removed using kits. The purity and quality of RNA samples were determined by measuring the ratio of absorbance at 260 and 280 nm and agarose gel electrophoresis. Detect the light absorbance at 260 to adjust the concentration of each RNA sample to make it consistent, and use the kit for one-step RT-PCR. Gene toy F is one of the key enzyme genes in the biosynthesis of toyF, using primers: toyF F: 5'-CTGTCGCTGGAGCTGGTGCG; toyF R: 5'-CAGCGACGAGGGCGCGGCGG. A 400 bp fragment of toyF gene can be amplified, and 16S rDNA was used as RT- Reference for PCR analysis. 16S rDNA F: 5’-CGATTACTAGCAACTCCGAC; 16S rDNA R: 5’-GGGGTGATGGGGACTCACAG. A 200 bp fragment o...
Embodiment 3
[0035] Example 3 : Verification of Fermentation Performance of Amylase Streptomyces Chromogenes Original Strain and Recombinant Strain
[0036] For the recombinant strain 1628-ADPA and the original strain S. diastatochromogenes 1628 conducted a 250 mL shake flask fermentation experiment, and studied the amylase chromogenic Streptomyces from the perspective of fermentation adp The overexpression of A gene was verified to increase the production of toyocamycin. The rotating speed of the reciprocating shaker was 200 r / min, 28°C, and fermented for 96 hours. The control group was the origin strain of Streptomyces chromogenes amylase. After the fermentation, the content of toyocamycin in the fermentation liquid was determined. As shown in Table 1, the yield of the recombinant toyocamycin was higher than that of the original strain throughout the fermentation process, and the final yield of the recombinant toyocamycin reached 177.62 mg / L, which was about 31.6% higher than that ...
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