Operon for synthesizing Pantoea agglomerans beta-carotene, expression vector and use thereof
A technology of carotene and Pantoea agglomerans, applied in the directions of application, introduction of foreign genetic material using vectors, biochemical equipment and methods, etc.
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Embodiment 1
[0055] Embodiment 1 strain separation
[0056] Get the discolored rice sample and use 70% (v / v) ethanol surface disinfection for 30 seconds, place it in a small mortar and grind after rinsing with sterile water, put tissue fluid in KB medium (2g / L peptone, 10g / L glycerol , 1.5g / L K 2 HPO 4 , 1.5g MgSO 4 ·7H 2 (0, 15g agar, pH 7.2) was separated by drawing a line, and after culturing at 28°C for 24 hours, a single colony was picked and purified. The picked yellow colonies are round, raised, smooth, moist, with neat edges, no fluorescent pigments, and the Gram staining reaction is negative; the cells are further screened under a microscope to show straight rods, perinatal flagella, and no spores strains. The obtained strain was diluted 10 4 After that, they were homozygous on KB solid plate medium repeatedly twice.
Embodiment 2
[0057] The extraction of embodiment 2 total DNA
[0058] The bacterial single strain that is isolated in embodiment 1 is cultivated 16 hours in 10ml liquid LB medium (5g / L yeast extract (Invitrogen), 5g / L NaCl, 10g / L tryptone, phosphate buffer pH7.5) , the cell culture solution was centrifuged at 6,000 g for 5 minutes to obtain the cell pellet. These pellets were first frozen at -20°C for 1 hr, then washed once with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and then 20 μl of sterile water with a concentration of lysozyme (Sigma-Aldrich) of 10 mg / mL was added Suspend and incubate on a shaking table at 37°C for 1 hr.
[0059] Then add 50 μl of 0.5M EDTA, 50 μl of 10% (w / v) SDS and 50 μl of 5M NaCl and shake gently to mix, then add 10 μl of 20 mg / mL protein kinase K (Takara Japan) , and the reaction was incubated at 37°C for 1 hr. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1, volume ratio) equivalent to the volume of the culture liquid (1 volume). Th...
Embodiment 3
[0061] Embodiment 3 Pantoea agglomerans strain homology analysis
[0062]Using the total DNA extracted in Example 2 as a template, the 16s rRNA-specific primers of Pantoea agglomerans were used for amplification. The amplification primers were P16SF: 5'GGTTACCTTGTTACGACTT-3' and P16SF: 5'-AGAGTTGATCCTGGCTCAG-3'. Using KOD Plus (Toyobo Japan) as Taq DNA polymerase, the amplification conditions were as follows: 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 120 seconds, and 30 cycles of amplification. After the cycle is over, add 2 units of rtaq enzyme (Bao Bioengineering (Dalian) Co., Ltd.), extend at 72°C for 300 seconds, and the length of the amplified fragment is 1500bp (see below, please supplement the sequence). After PCR, 1% (w / v) agarose gel was recovered, and 10 μl was directly connected to T / A carrier (Treasure Bioengineering (Dalian) Co., Ltd.), and connected overnight at 4°C.
[0063] The vector was first transformed into Escherichia coli DH5α competent cells, ...
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