Method for constructing high-expression trehalose synthase engineering bacteria using pcry3aa promoter
A high-expression technology of trehalose synthase, applied in the field of genetic engineering, can solve the problems of high extraction cost, difficult manufacturing, complicated process, etc.
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Embodiment 1
[0138] The Pcry3Aa promoter derived from Bacillus thuringiensis and the PhoD signal peptide of Bacillus subtilis were connected into a fragment Pcry3Aa-PhoD by overlapping PCR, which was constructed into the shuttle plasmid PHT01 to obtain the recombinant plasmid Pcry3Aa-PhoD-PHT01.
[0139] (i) extracting the genome of Bacillus thuringiensis LM79 thallus, using the genome as a template, performing PCR amplification to obtain the Pcry3Aa promoter fragment;
[0140] Described PCR primer sequence is as follows:
[0141] Upstream primer (Pcry3Aa-F): 5'- GGTACC GTGCTTTTTTTGTTGCAATT-3';
[0142] Downstream primer (Pcry3Aa-R): 5'-GACTGTCGTATGCCATTTTTCTTCCTCCCTTTCTTAT-3';
[0143] The underline is the enzyme cutting site KpnI;
[0144] Described PCR amplification system is as follows:
[0145] 2×Taq PCR MasterMix 25 μl, 2.5 μl upstream primer with a concentration of 10 μmol / L, 2.5 μl downstream primer with a concentration of 10 μmol / L, 2.5 μl template, ddH 2 O make up 50 μl;
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Embodiment 2
[0204] The gene fragment SpoIIID-1 derived from Bacillus subtilis 168 was combined with the Km from plasmid pBRNeo by overlapping PCR technique r Ligated into fragment SpoIIID-1-Km r , and then the fragment was electrotransformed into Bacillus subtilis WB800n after digestion with BamHI restriction enzyme to obtain the strain Bacillus subtilis WB800n [ΔSpoIIID] that does not express the SpoIIID gene.
[0205] (1) Using the DNA of the Bacillus subtilis 168 genome as a template, PCR amplification obtains the fragment SpoIIID-1; the nucleotide sequence of the SpoIIID-1 gene fragment is shown in SEQ ID NO.4;
[0206]The PCR amplification primer sequence of described fragment SpoIIID-1 is as follows:
[0207] Upstream primer (SpoIIID-1-F): 5'-CGC GGATCC GCGGGCGCTTTGGTTGATTTGAT-3';
[0208] Downstream primer (SpoIIID-1-R):
[0209] 5'-TCTTGTTCAATCATTGGAAACACCAAATTCCTTCGCAATGAC-3';
[0210] The single underline is the BamHI restriction site; the fragment SpoIIID-1 is the entire ...
Embodiment 3
[0251] The production of trehalose synthase, the operation steps are as follows:
[0252] 1) Preparation of the first-class species: transfer the high-expression trehalose synthase engineered bacteria WB800n[ΔSpoIIID]+[Pcry3Aa-PhoD-PHT01-TreS] prepared in Example 2 from the LB plate containing chloramphenicol 20ug / mL A single colony of the strain was cultured overnight in 3mL LB liquid medium at 37°C and 200rpm, and the obtained strain was a first-class species;
[0253] 2) Preparation of the secondary species: inoculate the primary species in 800 mL LB liquid medium containing 20 ug / mL of chloramphenicol, and cultivate to OD at 37°C and 200 rpm 600 is 0.9 (4.5 hours);
[0254] 3) Preparation of the tertiary species: Inoculate the second-level species into an 80L LB liquid fermenter at 37°C, control the pH to about 7.0 with citric acid and NaOH, ventilate and stir, control the dissolved oxygen at 20-30%, and cultivate to OD 600 is 0.9 (4.5 hours);
[0255] 4) Fermentation i...
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