A method for efficiently displaying trehalose synthase on the spore surface of Bacillus subtilis
A technology of Bacillus subtilis and trehalose synthase, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low display efficiency and cannot meet industrial application, etc., and achieves the effects of achieving genetic stability, improving efficiency and clear genetic background.
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Embodiment 1
[0087] Example 1: Construction of recombinant plasmids
[0088] (1) Cloning to obtain the spore capsid protein gene fragment
[0089] Using the Bacillus subtilis WB800n genome as a template, primers were designed for PCR amplification to obtain gene fragments of five spore capsid proteins;
[0090] The PCR amplification of described CotA protein gene sequence, primer nucleotide sequence is as follows:
[0091] cotA-F: 5'-aaaactggtctgatcggatccCGACCCGATATCCTGCCTTA-3'
[0092] cotA-R: 5'-gctgggtcatTTTATGGGGATCAGTTATATCCATCG-3'
[0093] The PCR reaction system is as follows:
[0094]2×Phanta Max Master Mix 25μL, 10μmol / L upstream primer cotA-F 2.5μL, 10μmol / L downstream primer cotA-R 2.5μL, template 2.5μL, use ddH 2 O supplemented to 50 μL;
[0095] The above-mentioned PCR reaction is carried out according to the following procedures:
[0096] Pre-denaturation at 95°C for 3min; denaturation at 95°C for 15s, annealing at 60°C for 15s, extension at 72°C for 45s, 30 cycles; ext...
Embodiment 2
[0228] Example 2: Preparation of Bacillus subtilis WB800n electroporation competent cells
[0229] Pick a single colony of Bacillus subtilis WB800n on the surface of fresh LB solid medium and culture it overnight in 5 mL LB medium. Take 1 mL of the overnight culture and insert it into 50 mL of GM medium (LB+0.5M sorbitol), and culture it with shaking at 37°C until OD 600 is 1.0. The bacterial solution was placed in an ice-water bath for 10 minutes, centrifuged at 5000 rpm at 4°C for 8 minutes, and the bacterial cells were collected. The cells were resuspended with 20 mL of pre-cooled ETM medium (0.5M sorbitol+0.5M mannitol+10% glycerol), centrifuged at 5000rpm at 4°C for 8min, the supernatant was removed, and washed three times in this way. Resuspend the washed bacteria in 500 μL of ETM medium, aliquot them into EP tubes, and aliquot 60 μL in each tube.
Embodiment 3
[0230] Embodiment 3: Into Bacillus subtilis WB800n with recombinant plasmid
[0231] Add 6 μL of the recombinant plasmid to 60 μL of competent cells, incubate on ice for 5 min, add it to a pre-cooled electroporation cuvette (2 mm), and perform electroporation at 2500 V for 5 ms. Immediately after the electric shock, add 1 mL of 37°C preheated RM medium (LB+0.5M sorbitol+0.38M mannitol) to the electroporation cup, revive and culture at 37°C for 3 hours, and spread on the LB plate containing spectinomycin superior. Inverted culture at 37°C, and strains resistant to spectinomycin were screened.
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