An engineering bacterium stably displaying trehalose synthase on the surface of spores and its construction method
A technology of trehalose synthase and trehalose, applied in the field of bioengineering, can solve the problems of insufficient recycling, poor stability and the like
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Embodiment 1
[0086] Example 1: Construction of gene knockout fragments
[0087] (i) extracting Bacillus Subtilis 168 DNA, using the DNA as a template, performing PCR amplification to obtain the homology arm sleB1;
[0088] Described PCR primer sequence is as follows:
[0089] sleB-up:5'-CGGGATCCCGGGGGATGATGTGGTCGAG-3';
[0090] sleB-down:5'-ACTGACTCACTCAAAAATAACCCCCGCTACT-3';
[0091] Described PCR amplification system is:
[0092]
[0093] The PCR amplification procedure is as follows:
[0094] Pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 1.5 min, 30 cycles; final extension at 72°C for 10 min, storage at 4°C;
[0095] The PCR product was checked by agarose gel electrophoresis, the length was 592bp, and the SanPrep column DNA gel recovery kit (Shanghai Sangong) was used for gel recovery, and the recovered product was stored at -20°C for later use;
[0096] (ii) extracting Bacillus Subtilis 168 DNA, using th...
Embodiment 2
[0141] Embodiment 2: prepare Bacillus Subtilis 168 competent state
[0142] (i) Pick a single colony of Bacillus Subtilis 168 on the surface of fresh LB solid medium, inoculate it in 10mL of GM medium, cultivate overnight at 37°C and 220r / min;
[0143] (ii) Take 1 mL of the above bacterial solution and transfer it to 100 mL of GM medium, and culture it to OD at 37°C and 220r / min 600 = 1.0;
[0144] (iii) Transfer the bacterial solution to a 100mL centrifuge tube and place in an ice bath for 10 minutes to stop the growth of the bacterial cells;
[0145] (iv) Centrifuge at 4°C, 7000r / min, 5min after ice bath, and collect the bacteria;
[0146] (v) The thalline after centrifugation is washed 2-3 times with pre-cooled electroporation buffer (ETM);
[0147] (vi) After washing, use 1000 μL electroporation buffer to resuspend the cells;
[0148] (vii) Aliquot the prepared competent cells into 100 μL tubes and store at -80°C for later use.
[0149] Among them, GM medium: LB mediu...
Embodiment 3
[0152] Example 3: sleB1-km r Electrotransformation of fragments into Bacillus Subtilis 168 and identification of positive recombinants
[0153] (i) sleB1-km r Fragments were digested with restriction endonuclease BamH I;
[0154] Enzyme digestion system (40μL) is as follows:
[0155]
[0156]
[0157] (ii) Concentrate and purify the digested product
[0158] (1) Add 1 / 10 volume of 3M sodium acetate and 2.5 volumes of absolute ethanol, and place in a -20°C refrigerator for 20 minutes;
[0159] (2) Centrifuge at 12000r / min for 5min to obtain a precipitate;
[0160] (3) 300 μL of 75% ethanol solution by volume to resuspend the precipitate;
[0161] (4) Centrifuge at 12000r / min for 5min, remove ethanol, and air-dry at 37°C for 30min;
[0162] (5) Add 15-18μL ddH 2 O Resuspend DNA and store at -20°C.
[0163] (iii) Electroconversion
[0164] Firstly, sleB1-km was measured by nucleic acid ultramicro spectrophotometer r The concentration of the fragments was 100-400ng...
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