A recombinant Escherichia coli overexpressing lsrb gene and its construction method and application
A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of genetic engineering, can solve the problems of reduced cell viability, non-reusable use, and increased operating costs, and achieve the effects of high fermentation yield, cost saving, and short cycle
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0057] Example 1: Molecular transformation of target strains
[0058] 1. Amplify the target gene
[0059] Using E. coli CIBTS1688 genome as a template, the target gene lsrB was amplified. Based on the NCBI gene sequence, the primers designed for amplification are shown in Table 1. Wherein, the primers contain XhoI and NcoI restriction sites for subsequent ligation with plasmid pET28a.
[0060] Table 1 Experimental primer sequences used for amplification
[0061]
[0062] PCR uses 25μL reaction system, primers lsrB-F, lsrB-R each 1.5μL; 1.0μL template (genome); 1.0μL KOD enzyme (DNA Polymerase 1.0unit / μL); 10μL dNTP (deoxyribonucleoside triphosphate) ) (150 mM); 10 μL of sterile water, respectively packed into a tube of 25 μL, recovered and purified by gel after PCR
[0063] 2. Construction of Recombinant Plasmids
[0064] 2.1 Plasmid extraction
[0065] (1) E. coli DH5α glycerol bacteria (containing plasmid pET28a) were inoculated into liquid LB (kana-resistant concen...
Embodiment 2
[0089] Example 2: Characterization experiment of biofilms
[0090] 1. Crystal violet staining
[0091] In this example, the crystal violet staining method was used to compare the relative expression of the biofilm of two target bacteria (the original strain WT: E. coli CIBTS1688, and the recombinant E. coli prepared in Example 1). Therefore, in 96-well plates, these two strains were verified for film formation.
[0092] (1) The 2 strains were cultured at 37° C. and 200 rpm to log phase (OD600 was about 0.6).
[0093] (2) Dilute the bacterial liquid to an OD600 of 0.1, add 180 μL of LB liquid medium to the 96-well plate, and then insert 20 μL of the diluted bacterial liquid into the medium, and culture at 37°C for 6h, 12h, and 18h respectively. , 24h, 30h, 36h, 42h to make Escherichia coli form a film at the bottom of the 96-well plate.
[0094] (3) Pour off the LB liquid medium, rinse with PBS for 2-3 times, fix the biofilm with methanol for 15 minutes, pour out the metha...
Embodiment 3
[0105] Example 3: Study on the performance of L-threonine produced by fermentation of the target strain
[0106] 1. Free fermentation
[0107] (1) Restoring strain activity
[0108] Take the bacteria preservation tubes of the original bacteria and the recombinant bacteria, respectively, and place them in an ice box to wait for thawing. After thawing, take an appropriate amount of bacterial liquid into the LB shake tube, and cultivate at a constant temperature of 37 °C.
[0109] (2) Connect the fermentation broth
[0110] (i) Add 1 mL of the revived bacterial solution to a 100 mL shake flask, and place it into a 37°C incubator for constant temperature incubation for 12 hours.
[0111] (ii) Pour 100mL fermentation medium into a 500mL conical flask, take 10mL of the bacterial liquid cultured in step (1) and insert it into the conical flask, carry out constant temperature fermentation in a shaker at 37°C, and detect the fermentation broth every 6 hours The residual glucose amou...
PUM
| Property | Measurement | Unit |
|---|---|---|
| density | aaaaa | aaaaa |
| pore size | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More