Shewanella strain metabolism acetate transformation method and application
A technology of Shewanella and acetate, which is applied in the field of genetic engineering and biological metabolism, can solve the problems of large consumption, limited production, and inability to metabolize acetic acid, etc., to enhance power generation performance, increase power density, and increase extracellular electrons. The effect of transmission efficiency
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Embodiment 1
[0034] Example 1: Construction of recombinant plasmids.
[0035] Synthesis and optimization of genes Ato1, Ato2 and GltA for metabolism of acetate uptake by Shewanella. Using the Biobrick method, the above genes were ligated by restriction enzymes to construct recombinant plasmid pAU3.
[0036] ①Add an XbaI restriction site to the 5' end of the synthesized target gene fragment, and add SpeI and SbfI restriction sites to the 3' end in sequence. First utilize XbaI and SbfI enzyme (well-known, commercially available) to gene Ato-1 enzyme cut (nucleotide sequence is SEQID NO.1), utilize SpeI and SbfI enzyme (well-known, commercially available) to self-contained The pYYDT plasmid at the restriction site was subjected to restriction restriction (the nucleotide sequence is SEQ ID NO.4). Since the XbaI and SpeI enzymes are homologous enzymes, the same cohesive ends will be produced after digestion, and then ligated by T4 ligase to construct a recombinant plasmid containing the gene ...
Embodiment 2
[0040] Example 2: Construction of recombinant Shewanella strain.
[0041] Such as figure 2 As shown, it is a schematic diagram of engineering Shewanella acetate metabolism design constructed by the present invention, including the whole process from promoting acetate uptake into cells to promoting TCA cycle and intracellular metabolism, specifically obtaining a strain capable of Engineering Shewanella bacteria for glucose metabolism.
[0042] Transformation: Take out 100 μL of E.coli WM3064 competent cells from the -80°C refrigerator, place them in an ice box for natural thawing, add 10 μL of recombinant plasmid pAU3, let stand on ice for 30 minutes, heat shock at 42°C for 90 seconds, and then stand on ice for 2- 3min, add 1mL LB+DAP liquid medium (5g / L yeast extract, 10g / L tryptone, 10g / L NaCl, 0.059g / L 2,6-diaminopimelic acid) into the Ep tube, place Recover in a shaker at 37°C and 220 rpm for 1 hour. After centrifugation, apply to LB+DAP+Kan plate (5g / L yeast extract, 1...
Embodiment 3
[0048] Example 3: The recombinant Shewanella engineering bacterium Ace-3 was fermented in SBM liquid culture solution containing 10 mM acetate solution (including aerobic and anaerobic).
[0049] To determine cell growth, 2 mL of culture suspension of WT, Ace-3 strain was inoculated into 100 mL of SBM medium. The cell culture was cultured on a shaker at 30°C and 200 rpm, and samples were taken regularly to measure the cell density (optical density at 600 nm, ie OD 600 ), measured with a UV-Vis spectrophotometer (TU-1810, Beijing, China).
[0050] Both WT and Ace-3 strains were cultured overnight at 30°C and 200 rpm in 10 mL of LB medium.
[0051] A 3% inoculum of each suspension was transferred into SBM (pH 7.2) supplemented with 10 mM acetate as substrate. When needed, supplemented with 50 mg / mL kanamycin and 0.75 mM IPTG.
[0052] Metabolites in shake flasks were analyzed by a high-performance liquid chromatography (HPLC) system equipped with UV detection. All fermentati...
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