Bacterial cellulose producing bacterial strain and construction method and application thereof
A bacterial cellulose and a technology for producing strains, applied in the field of genetic engineering, can solve problems such as low yield and long fermentation time, and achieve the effects of increased yield, shortened fermentation time, and increased yield
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[0024] Example 1: Cloning of Acetobacter xylinum cellulose synthase subunit bcsB gene
[0025] Firstly, the subunit (bcsB) related to bacterial cellulose secretion in the cellulose synthase of Acetobacter xylinum was amplified and sequenced, and a 470bp DNA fragment was obtained after amplification. The specific process is as follows:
[0026] First, extract the genome of Acetobacter xylinum ATCC700178, design primer pairs bcsB-FA, bcsB-RC, and use the extracted genome of Acetobacter xylinum ATCC 700178 as a template for PCR amplification. The PCR reaction system is 5×PrimeSTARbuffer(Mg 2+ ) 20.0μl, dNTP Mixture (2mM) 10.0μl, bcsB-FA 1.0μl, bcsB-RC 1.0μl, PrimeSTAR 1.0μl, template 0.5μl; fill up to 100.0μl with sterile water, and then divide it into 25.0μl / tube. Reaction conditions: 95°C for 5min; 95°C for 10sec; 60°C for 30sec; 72°C for 3minc; 72°C for 10min; 35 cycles, the amplified PCR product was purified by a gel recovery kit.
[0027] The primers required for the above are ...
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[0030] Example 2: Construction and verification of recombinant plasmid pSA19-bcsB.
[0031] The plasmid fragment pAH4 was synthesized according to the endogenous plasmid sequence of Acetobacter xylinum on NCBI, and the plasmid pAH4 and plasmid pUC18 were single-enzyme digested with hindIII and recombined to obtain the ampicillin-resistant recombinant plasmid pSA19. The pSA19 plasmid was digested with EcoRI and SalI and recovered by gel. The target fragment recovered from the above gel was connected to the pSA19 plasmid through one-step cloning, thereby constructing the vector pSA19-bcsB with ampicillin resistance as the selection marker. Then the pSA19-bcsB vector was verified and double-enzyme digestion verification was performed. The verification results were consistent with expectations. The plasmids with the double-enzyme digestion results consistent with expectations were sent for sequencing and consistent with expectations, proving that the vector was successfully construct...
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[0032] Example 3: Construction and molecular verification of the transformant of Acetobacter xylinum 700178 transformed by the pSA19-bcsB vector.
[0033] 1μg of pSA19-bcsB vector was transformed into Acetobacter xylinum ATCC700178, and the transformation process used electrotransformation method. The transformants were screened on a selective medium containing ampicillin resistance, and finally a genetically stable Acetobacter xylinum bcsB transformant was obtained. PCR verification proved that the above transformants were successfully inserted into the pSA19-bcsB plasmid.
[0034] The specific method of the above electroconversion is as follows:
[0035] Competent preparation:
[0036] 1. Scrape a ring of Acetobacter xylinum seeds from the plate into a 500ml Erlenmeyer flask containing 100ml seed liquid, and add the cellulase for sterilization to make the amount of cellulase per ml of culture medium 0.5U. 30°C, 150rpm, culture for 18h, so that the OD value of the bacterial solutio...
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