A protein that promotes the production of poly-3-hydroxybutyrate by Alcaligenes rosenbergii and its application
The technology of Alcaligenes eutropha and hydroxybutyrate is applied in the fields of biological materials and microbial metabolic engineering, and can solve the problems of high production cost, high cost, and failure to improve the production capacity of the strain, and achieve the effect of improving the growth rate of the strain
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Embodiment 1
[0046] This example amplifies the gene encoding the protein that promotes the production of poly-3-hydroxybutyrate by Alcaligenes rosenbergii.
[0047] To prepare LB liquid medium, add 5 g / L yeast extract powder, 10 g / L tryptone and 10 g / L NaCl to the water, stir evenly, adjust the pH to 7.0, and then autoclave at 121 °C for 20 min. Add 1.5% agar to LB liquid medium by mass percentage to get LB solid medium. Cultivate Alcaligenes eutropha H16 in LB liquid medium (30°C, 200 rpm) Cupriavidus necator H16, purchased from the German Collection of Microorganisms and Cell Cultures), and then extract genomic DNA, using genomic DNA as a template, with upstream primer 5'-TCAGGCAGCCGTCGTCTTCT-3' (Primer F) and downstream primer 5'-ATGATCCTCACCCCGGAAC -3' (Primer R) PCR amplification of the gene encoding the protein that promotes the production of poly-3-hydroxybutyrate by Alcaligenes rosenbergii (sequence shown in SEQ ID NO.2), the PCR reaction system is shown in the table 1, the PCR r...
Embodiment 2
[0051] In this example, the PCR product obtained in Example 1 was purified by agarose gel electrophoresis.
[0052] Weigh 0.2 g of agarose and add it to 20 mL of 1×TEA solution and heat to dissolve, then add 1 μL of nucleic acid dye, mix well and pour it into the tank to make gel, to obtain agarose gel, add 10 to 50 μL of the PCR product After mixing μL of loading buffer, it was added to the sample well of agarose gel, and the corresponding DNA marker was added, the voltage was 121 V, the current was 200 mA, and the electrophoresis was performed for 35 min. The electrophoresis was observed with a gel imager. The DNA fragments were recorded, recovered and purified, and the concentration was detected to obtain the coding gene of the protein that promotes the production of poly-3-hydroxybutyrate by Alcaligenes rosenbergii.
Embodiment 3
[0054] In this example, an expression vector containing the gene encoding the protein that promotes the production of poly-3-hydroxybutyrate by Alcaligenes rosenbergii was constructed.
[0055] restriction endonuclease Sal I digested the vector pBBR1MCS2 and purified it, then performed Gibson assembly on the purified enzyme digested reaction product and the gene fragment purified in Example 2. , Transform the reaction product into Escherichia coli Dh5α: place the competent Escherichia coli Dh5α on ice to melt, add 10 μL of the LGibson assembly product to the melted competent cells and mix evenly, ice-bath for 30 minutes, and place the mixture in a 42°C water bath Incubate for 90 s, then place in ice for 2 min, add 500 μL LB liquid medium, incubate at 37 °C, 200 rpm for 45 min, take 100 μL of the recovered bacterial solution and spread it on the LB solid plate with corresponding resistance , cultivated at 37°C for 16 hours, picked a single colony to verify the recombinant bac...
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