Construction of recombinant strain capable of producing arginine deiminase and directional modification method thereof
An arginine deiminase and a construction method technology, which can be applied in the directions of microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., and can solve problems such as limiting the application prospects of recombinant ADI enzymes.
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Embodiment 1
[0023] Embodiment 1 Construction of recombinant ADI
[0024] According to the gene sequence of the ADI, design primers:
[0025] F: 5'-GCT CATATG TCCGCTGAAAAACAGAAGTACG-3’ ( Nde I)
[0026] R: 5'-AT CTCGAG TTAGTAGTTGATCGGGTCGCGCA -3' ( xho I)
[0027] Introduce upstream and downstream separately Nde I and xho I enzyme cutting site (underlined), the primers used in the present invention were all synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.
[0028] Using Pseudomonas mutans cells as a template, the ADI gene fragment was amplified by PCR, and the reaction system was 50 μL, as shown in Table 1:
[0029] Table 1 PCR amplification reaction system of ADI gene fragment
[0030] 10×Ex Taq Buffer
[0031] Reaction program: Denaturation at 94 °C for 10 min; reaction at 94 °C for 1 min, 56 °C for 45 s, and 72 °C for 2 min for 30 cycles, followed by extension at 72 °C for 10 min to obtain the PCR product ADI.
[0032]The PCR product A...
Embodiment 3
[0051] Example 3 Screening of ADI mutant library
[0052] A single colony in the ADI mutant library was transferred to an LB / Kan plate containing IPTG (final concentration 0.2 mmol / L), and cultured at 30°C until a single colony grew.
[0053] According to the reaction that ADI catalyzes arginine to produce citrulline and ammonia, a 96-well plate screening method for mutants with ADI activity was designed. Specific operation: Take a 96-well plate, first add 50 μl of 0.2mol / L phosphate buffer solution (pH 7.4) to each well, pick a single colony induced by IPTG and mix it in the well (the sample without bacteria is used as a control), Then add 50 μl of 1 mmol / L L-arginine hydrochloride, 0.2 mol / L phosphate buffer (pH 7.4), react at 37°C for 15 min, add 90 μl of mixed acid to terminate the reaction, and then add 30 μl of diacetylmonoxime-sulfur Amidurea solution, mix well, react at 37°C for 2 hours, measure OD 530 . The reaction solution in the well where the strain with ADI ac...
Embodiment 4
[0054] Example 4 ADI mutant strain M314
[0055] A single colony of the ADI mutant strain M314 was inserted into LB / Kan liquid medium and cultured overnight at 37°C and 200r / min. Bacteria were collected, plasmids were extracted, and purified. The plasmid was sequenced by Shanghai Sangon Bioengineering Technology Service Co., Ltd. See SEQ ID NO: 1 in the sequence table. It was determined that M314 carried three mutation sites, namely A128T, H404R and I410L.
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