Spac was inserted in to the plasmid pUB-Tet, in addition to a new replication-thermosensitive plasmid, pUB-MazF, was constructed (Fig. 1d).Functional Verification of pUB-MazF and pUB -EXThe replication of pUB-MazF was verified in B. subtilis WB600 and B. licheniformis BL-109 (Fig. 3a), respectively. After 48 hr cultivation in LB medium with no 1 mmol/l IPTG at 30 and 42 , the recombinant B. subtilis and B. licheniformis harboring pUB-MazF grew effectively on LB plates with 15 and 2 g/ml tetracycline, respectively. After 48 hr cultivation in LB medium with 1 mmol/l IPTG at 42 , no recombinants grew on LB plates with tetracycline (Fig. 3a). Theseresults indicated that plasmid pUB-MazF could possibly be substantially cured by induction of MazF with IPTG. The replication and integration of pUB -EX1 using the help of pUB-MazF was verified in B. subtilis WB600 and B. licheniformis BL-109 (Fig. 3b). Each B. subtilis WB600 and B. licheniformis BL-109 harboring pUB-MazF and pUB -EX1 had been grown properly on LB plates complemented with two g/ml tetracycline and 20 g/ml kanamycin at 30 , which indicated that pUB-MazF assisted with the replication of pUB -EX1 and both plasmids may very well be maintained and replicated in each hosts at reduced incubation temperature. By incubating the strains in LB medium with 1 mmol/l IPTG at 42 and 200 rpm for 48 hr, each strains grew properly on LB plates with 20 g/ml kanamycin but not on two g/ml tetracycline plates (Fig. 3b). The results suggested that pUB -EX1 was integrated in to the chromosome with choice stress (20 g/ml kanamycin) and pUB-MazF was lost (curing) as a result of expression of MazF in pUBMazF.Overexpression of Thermophilic -Amylase in B. licheniformis Applying pUB-MazF and pUB -EXRecombinant B. licheniformis strains overexpressing -amylase have been created as described within the following measures (Fig. 4).| Journal of Industrial Microbiology and Biotechnology, 2022, Vol. 49, No. three reached 50 753 U/ml following 72 hr of cultivation and then decreased slightly. Right after fermentation, the principle protein within the broth was the expressed -amylase within the SDS-PAGE profile (band at 55.5 kDa) (Fig. 6b). The yield on the -amylase was 22-fold much better than a previous report (Chen et al., 2015). The recombinant BLiS-002 showed fantastic genetic stability and all the colonies with the recombinant BLiS-002 in fermentation broth could kind a halo zone on starch plates right after 80 hr fermentation (Fig. S2). By using this newly created strategy, we’ve got effectively and promptly overexpressed several other industrial enzymes, which includes pullulanase, lactase, bacterial -amylase and alkaline protease in B.PDGF-BB Protein Species licheniformis (data not shown).GM-CSF Protein Source Step 1: pUB-MazF was transformed into B.PMID:25040798 licheniformis BL-109 by electroporation. Right after transformation, pUB-MazF was integrated into the chromosome of B. licheniformis BL-109 by raising the temperature to 42 and choosing for resistance to tetracycline. The resulting recombinant was named as B. licheniformis BL-UBM. Step two: the -amylase integrative expression plasmid pUB -amyS (Fig. 1e) was constructed and transferred into B. licheniformis BLUBM by electroporation. The resulting transformant was named as BL-amyS. Production of -amylase was predetected on starch plates containing two g/ml tetracycline and 20 g/ml kanamycin at 30 . Step 3: BL-amyS was cultivated in LB medium with 500 g/ml kanamycin and 1 mmol/l IPTG at 42 for 4 subculture cycles, top to the integrated fragment of helper plasmid pUBMazF becoming cured from the chromosome and there becoming no additional.