Method for preparing fat alkane and intermediate alkene
A technology of olefins and pentadecane, applied in the direction of microorganism-based methods, biochemical equipment and methods, hydrogenation hydrocarbon production, etc., can solve the problems of many by-products and low product yields
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Embodiment 1
[0137] The following plasmids were constructed for the introduction of foreign nucleic acid sequences in BL21(DE3) host cells. All cloned genes were under the control of an IPTG-inducible promoter (T7, tac or lac promoter).
[0138] In this example, the polyketide synthase gene sgcE (SEQ ID NO: 1) was introduced. The polyketide synthase gene sgcE expresses the protein polyketide synthase SgcE (SEQ ID NO: 5). The exogenous polyketide synthase interacts with the enol-ACP-reductase FabI (SEQ ID NO: 6) expressed from the enol-ACP-reductase gene fabI (SEQ ID NO: 2) to synthesize pentadecane.
[0139] According to the standard molecular biology method, the cloning of the gene and the construction of the vector, in short, include the following steps:
[0140] The sgcE gene was cloned on pET28a. Plasmid Plasmid pWK1 ( figure 1 ) is obtained by cloning the sgcE gene into the pET28a vector through BamH I and HindIII. The constructed plasmid pWK1 was transformed into Escherichia col...
Embodiment 2
[0141] Example 2, Production of Hydrocarbons as Biofuels
[0142] The Escherichia coli WK1 seed solution cultivated overnight at 37 degrees Celsius was diluted into 200 ml of LB medium at a volume ratio of 1:100; after culturing at 37 degrees Celsius for 3 hours, when the OD600 of Escherichia coli WK1 was 0.4-0.6, add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.1mM; after adding IPTG for 15-20 hours of induction, extract the bacterial liquid with ethyl acetate, recover the organic phase and dry the solvent by rotary evaporation to 1-2ml. After the samples were filtered through a 0.22 μm filter membrane, they were detected by GC-MS.
[0143] Experimental results: Biodiesel n-pentadecane C can be clearly detected in the sediment of Escherichia coli WK1 15 h 32 ( Image 6 ). It shows that alkane compounds can be obtained efficiently by introducing sgcE into Escherichia coli.
Embodiment 3
[0145] In this example, polyketide synthase gene sgcE (SEQ ID NO: 1) and thioesterase gene sgcE10 (SEQ ID NO: 3) were introduced. The proteins expressed by the polyketide synthase gene sgcE and the thioesterase gene sgcE10 are polyketide synthase SgcE (SEQ ID NO: 5) and thioesterase SgcE10 (SEQ ID NO: 7), respectively.
[0146] According to the standard molecular biology method, the cloning of the gene and the construction of the vector, in short, include the following steps:
[0147] The sgcE10 gene was cloned into pET28a. Plasmid pWK2 ( figure 2 ) is obtained by cloning the sgcE10 gene into the pET28a vector through Nde I and XhoI. The sgcE and sgcE10 genes were cloned in series on the pET28a vector. Digest the plasmid pWK2 with SpeI and Xho I, and recover the linear DNA fragment; digest the plasmid pWK1 with Xba I and Xho I, recover the sgcE gene fragment; connect the sgcE gene fragment and the pWK2 vector fragment, transform into Escherichia coli, and pass the card Na...
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