A citrate transporter and its application in lipid synthesis
A technology for transporting protein and citric acid, which is applied in the fields of genetic engineering and microbial engineering to achieve the effect of promoting the production of fatty acids by microorganisms and improving the biosynthetic ability
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Embodiment 1
[0040] Example 1: Screening of genes encoding citrate-ketoglutarate transporters
[0041] Specific steps are as follows:
[0042] Using the YALI0 lipolytic citrate-ketoglutarate transporter gene sequence identified in NCBI (NCBI ID: YALI0B10736p) as a template, BLAST comparison was performed in the gene bank of the sequenced M.alpina ATCC 32222 strain According to the condition of homology>30%, an alternative target gene sequence (MA-00120-22) was obtained; the final target gene was named MaYHM (nucleotide sequence as shown in SEQ ID No.2), The corresponding protein is named MaYHM (the amino acid sequence is shown in SEQ ID No.1).
[0043] The full-length corresponding cDNA of MaYHM is 930bp, encoding 309 amino acids. In order to further judge whether the screened MaYHM belongs to the citrate-ketoglutarate transporter, amino acid homology and conservative structure analysis were carried out between it and the citrate-ketoglutarate transporter identified in NCBI.
[0044] Su...
Embodiment 2
[0045] Example 2: Cloning of MaYHM
[0046] Specific steps are as follows:
[0047] The total RNA of Mortierella alpina (Mortierella alpina) ATCC 32222 was extracted using the TRIzol method, and cDNA was obtained by reverse transcription according to the instructions of the Thermo reverse transcription kit, and amplified by PCR reaction in the cDNA library of Mortierella alpina (Mortierella alpina) ATCC 32222. Add MaYHM, and the primers used to amplify MaYHM are listed in Table 1.
[0048] The PCR instrument used is BIO-RAD T100 Thermal Cycler, using KOD plus high-fidelity DNA polymerase, the reaction system is 50 μL, and the content of the system is carried out according to the instructions of the DNA polymerase; the reaction process is as follows: pre-denaturation at 95°C for 5 minutes, then denaturation at 95°C 30s, annealing at 55°C for 30s, extension at 68°C for 1.5min, repeat the above three steps 30 times, then fully extend at 68°C for 5min, and finally drop to 12°C fo...
Embodiment 3
[0052] Embodiment 3: Construction of overexpressing MaYHM recombinant bacteria
[0053] Specific steps are as follows:
[0054] (1) Construction of Mortierella alpina expression vector
[0055] The MaYHMDNA fragment obtained in Example 2 and the expression vector pBIG2-ura5s-ITs were digested using restriction endonucleases XhoI and KpnI, and then utilized T 4 Ligase ligated the digested and purified MaYHM DNA fragment with pBIG2-ura5s-ITs to obtain a ligation product. The specific enzyme digestion system (20 μL) is shown in Table 2.
[0056] Table 2 enzyme digestion system
[0057] Reagent Dosage 10×cutmart buffer 2μL restriction endonuclease 1μL PCR product or vector 200ng~1μg wxya 2 o
Make up to 20μL
[0058] After connecting the obtained ligation product overnight at 4-16°C, transform it into Escherichia coli TOP10 competent cells. The transformation method is as follows: take 100 μL of competent cells in a sterile state, ...
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