Engineering bacteria for degrading pyrene gene efficiently and construction thereof
A technology of genetically engineered bacteria and construction methods, applied in the field of pyrene-degrading genetically engineered bacteria and its construction, can solve the problems of microorganisms lacking monooxygenase genes and monooxygenase cannot be expressed, and achieve important application value and broad market prospects Effect
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Embodiment 1
[0045] (1) Extraction of total RNA
[0046] 1) Cultivate the white rot fungus with potato medium to the logarithmic growth phase, take 50 mg of the fungus, add 500 μL of denaturing solution and 3.6 μL of β-mercaptoethanol, mix well, and homogenize with a homogenizing popsicle bath.
[0047] 2). Immediately add 100 μL sodium acetate (2mol / L, pH 4.0), 200 μL water-saturated phenol and 200 μL chloroform / isoamyl alcohol (24:1). After adding each component, cover the cap of the centrifuge tube, shake it gently to mix well, and put it in an ice bath for 15 minutes.
[0048] 3). Centrifuge at 10,000 rpm for 10 min at 4°C, and transfer the RNA-containing aqueous phase into a new centrifuge tube.
[0049] 4). Add isopropanol equal to the volume of the extract, mix well, and precipitate RNA at -20°C for 1 hour or longer. Centrifuge at 15000rpm for 15min at 4°C
[0050] 5). Gently decant the isopropanol, add 100 μl to dissolve the RNA particles, and add an equal volume of isopropanol to...
Embodiment 2
[0104] (1) Extraction of total RNA
[0105] 1) Cultivate the white rot fungus with potato medium to the logarithmic growth phase, and take 100 mg of the fungus
[0106] Mix 500 μL of denaturing solution and 3.6 μL of β-mercaptoethanol, and homogenize with a homogenizer popsicle bath.
[0107] 2). Immediately add 100 μL sodium acetate (2mol / L, pH 4.0), 200 μL water-saturated phenol and 200 μL chloroform / isoamyl alcohol (24:1). After adding each component, cover the cap of the centrifuge tube, shake it gently to mix well, and put it in an ice bath for 15 minutes.
[0108] 3). Centrifuge at 10,000 rpm for 10 min at 4°C, and transfer the RNA-containing aqueous phase into a new centrifuge tube.
[0109] 4). Add isopropanol equal to the volume of the extract, mix well, and precipitate RNA at -20°C for 1 hour or longer. Centrifuge at 15000rpm for 15min at 4°C
[0110] 5). Gently decant the isopropanol, add 100 μl to dissolve the RNA particles, and add an equal volume of isopropan...
Embodiment 3
[0164] (1) Extraction of total RNA
[0165] 1) Cultivate the white rot fungus with potato medium to the logarithmic growth phase, and take 80 mg of the fungus
[0166] Mix 500 μL denaturing solution and 3.6 μL β-mercaptoethanol, and homogenize with a homogenizer popsicle bath.
[0167] 2). Immediately add 100 μL sodium acetate (2mol / L, pH 4.0), 200 μL water-saturated phenol and 200 μL chloroform / isoamyl alcohol (24:1). After adding each component, cover the cap of the centrifuge tube, shake it gently to mix well, and put it in an ice bath for 15 minutes.
[0168] 3). Centrifuge at 10,000 rpm for 10 min at 4°C, and transfer the RNA-containing aqueous phase into a new centrifuge tube.
[0169] 4). Add isopropanol equal to the volume of the extract, mix well, and precipitate RNA at -20°C for 1 hour or longer. Centrifuge at 15000rpm for 15min at 4°C
[0170] 5). Gently decant the isopropanol, add 100 μl to dissolve the RNA particles, and add an equal volume of isopropanol to p...
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