Genetic engineering strain utilize sucrose to produce succinic acid from and method for production of succinic acid by fermenting the same
A genetically engineered bacteria and succinic acid-producing technology, applied in the field of bioengineering, can solve the problems of lack of sucrose utilization genes, inability to utilize sucrose, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] This example illustrates the method for constructing E. coli BA500. Expression plasmid expressing exogenous sucrose permease, sucrose hydrolase and fructokinase genes.
[0042] (1) Using LB medium, cultivate Escherichia coli AFP111 to OD at 37°C under aerobic conditions 600 =0.5~0.6, using CaCl 2 Preparation of Escherichia coli AFP111 competent strain lacking lactate dehydrogenase gene (ldhA), pyruvate formate lyase gene (pflB) activity and ptsG gene chromosome spontaneous mutation of phosphotransferase system;
[0043] The formula of LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 5g / L.
[0044] (2) Synthesize primers without restriction sites,
[0045] Upstream primer: 5'-CCGGTTGAGGGATATAGAGCTATCGAC-3';
[0046] Downstream primer: 5'-CTGTTGATCCGTTGTTCCACCTGAT-3'.
[0047] Extract the E.coli W genome, and use the E.coli W genome as a template to amplify the target gene fragment by PCR. The specific reaction conditions are: 94°C, 10min; (94°C for 45s, 60°C for...
Embodiment 2
[0052] This example illustrates a method for continuously cultivating and acclimating Escherichia coli (Escherichia coli) BA500.
[0053] The Escherichia coli BA500 constructed in Example 1 was used as the starting strain. Transfer 1% (v / v) inoculum from the cryopreservation tube into the test tube, incubate overnight at 37°C, 200rpm, then transfer 1% (v / v) inoculum into the Erlenmeyer flask, incubate at 37°C, 200rpm for 6-8h Obtain the bacterium liquid of logarithmic growth phase; The bacterium liquid of logarithmic growth phase is inoculated in the 500mL continuous culture device that 300mL fermentation medium is housed with the inoculum size of 10% (v / v), 37 ℃ of water bath heating, pass into Filter-sterilized CO 2 Maintain the anaerobic environment, and feed fresh fermentation medium into the culture device at a rate of 1.5 mL / h. Regularly take samples to detect the density of bacteria in the culture device, when the density of bacteria in the reactor reaches OD 600 =2~...
Embodiment 3
[0057] This example illustrates the method for screening and obtaining excellent Escherichia coli (Escherichia coli) BA501. Screening steps:
[0058] 1. Solid plate primary screening
[0059] Under aseptic conditions, take out 2-4mL bacterial liquid from the continuous culture device and dilute it with sterile water 1×10 4 Take 100 μL and spread it on a solid plate containing 2% sucrose, incubate at 37°C for 12 hours, and select a single colony of the mutant strain with a fast growth rate and relatively plump.
[0060] 2. Solid flat plate re-screening
[0061] The screened mutant strains were repeatedly transferred and cultured on the plate, and finally the strains BA501, BA516 were obtained, and BA528 showed strong growth rate and growth stability.
[0062] 3. Shake flask fermentation screening
[0063] The mutant strains BA500, BA501, BA516 and BA528 were cultured in the seed medium, 37°C, 200r / min, cultured for 12h, and then inserted into the Erlenmeyer flask containing...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com