A key gene for cyanobacterial aliphatic hydrocarbon synthesis and its application
A technology of aliphatic hydrocarbons and hydrocarbon genes, applied in bacteria, applications, genetic engineering, etc., can solve problems that have not reached industrial applications, and achieve the effect of increasing carbon emissions
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Embodiment 1
[0104] Embodiment 1: the cultivation and biomass determination of cyanobacteria
[0105] Cultivation and physiological index determination of cyanobacteria:
[0106] The cyanobacterial strains in Table 1 were cultured by aeration culture, and the algae strains were inoculated into a 500mL Erlenmeyer flask containing 300mL BG11 medium, at 30°C, 30μE.m -2 .s -1 Grow in continuous light for 8 to 10 days. In order to measure its growth, 10ml of the well-mixed cyanobacteria suspension was passed through a 0.45μm pre-weighed nitrocellulose membrane (ddH 2 O washed three times, 110 ℃ high temperature drying) after suction filtration, put 110 ℃, dry 24 hours, and weigh its dry weight. Finally, the dry weight of the bacteria was used as a reference index to measure the biomass. The strains and their sources are listed in Table 1.
[0107] Algae (bacteria) strains used in Table 1 and their sources
[0108]
[0109] a FACHB: Freshwater Algal Culture Collection of The Institute...
Embodiment 2
[0111] Embodiment 2: the aliphatic hydrocarbon composition and content determination of cyanobacteria
[0112] Each of the above-mentioned 200mL cyanobacteria (in Table 1) that was in the plateau phase and well mixed was collected, and mixed with 10mL ddH 2O resuspended the above-mentioned cyanobacteria respectively, and then placed them on an ice-water bath, and ultrasonically disrupted them for 10 minutes (10s on, 10s off). After crushing, add 30 μg of n-eicosane as an internal standard, and after mixing, add 10 mL of chloroform:methanol (V:V is 2:1), shake and mix vigorously for 1-2 hours. Then centrifuge at 6,000 g for 10 min, transfer the lower organic phase to a new test tube, and dry it with nitrogen at 55°C. Add 1ml of n-hexane to dissolve the extracted aliphatic hydrocarbons, then filter through a 0.22μm membrane filter, and transfer to a 2ml gas phase vial. Use Agilent7890A gas chromatography-mass spectrometry (GC-MS) to measure aliphatic hydrocarbons. The GC-MS te...
Embodiment 3
[0119] Example 3: Sequencing of the cyanobacteria genome and sequence retrieval of the key gene ado-aar for hydrocarbon production
[0120] (1) The extraction steps of the cyanobacteria genome are as follows:
[0121] Collect about 50ml of each bacterial solution that is well mixed, centrifuge at 5,000g, and use 1.8ml of solution A (50mM Tris-Cl+50mM Na 2 EDTA+1M NaCl), and then moderately homogenized with a 2ml tissue homogenizer to disperse the filamentous cell homogenate into a single cell state.
[0122] Then proceed as follows: the cell suspension of each of the above-mentioned cyanobacteria and algal strains is divided into 6 Eppendorf tubes (300 μl / tube), and 10% sodium lauryl sarcosine aqueous solution (Sarkosyl solution) is added to each tube. The final concentration is 0.1%, and then stored at 4°C for 1 hr, centrifuged at 10,000g for 15 min to pellet the cells; wash the cells twice with 1ml solution A;
[0123] After washing, resuspend the cyanobacteria and algae c...
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