Fig. 1 The flowchart for building of new plasmids. The kanamycin resistance cassette of parent plasmid pUB-EX (a) was removed by inverse PCR working with primers P1 and P2. The resulting fragment was ligated with a tetracycline resistance cassette fragment isolated from pHY300PLK by PCR employing primers P3 and P4 to yield helper plasmid pUB-Tet (b). The repF encoding a replicase in pUB-EX was disrupted by inverse PCR employing primers P5 and P6 plus the item was self-ligated to yield an intermediate plasmid pUB -sint. A downstream fragment of amyL, three -amyL , was amplified from B. licheniformis CBBD302 chromosome making use of primers P9 and P10. It was inserted into the BamHI web-site of pUB -sint as a homologous arm to yield integrative expression plasmid pUB -EX1 (c). The mazF cassette was recovered in the chromosome of B. subtilis TMO310 by PCR amplification applying primers P7 and P8 and cloned into the SmaI web-site of pUB-Tet to get helper plasmid pUB-MazF (d). The amyS encoding the thermophilic -amylase from G.IFN-gamma Protein custom synthesis stearothermophilus ATCC 31195 was isolated by PCR amplification with primers P11 and P12, along with the PCR product was cloned in to the XbaI and SmaI websites of pUB -EX1 immediately after getting digested by XbaI, yielding -amylase integrative expression plasmid pUB -amyS (e).MCP-1/CCL2 Protein Storage & Stability The elements in the vectors as follows: repF, encoding a thermosensitive replicase, repF, represents the nonfunctional thermosensitive replicase gene sequence; PamyL , SamyL , and Tart represent the promoter, signal peptide, and terminator kind pHY-WZX; and ori-T represents the temperature-sensitive replication origin.on the plasmids. The genome of B. subtilis TMO310 was utilised as the template for amplification with the isopropyl-1-thio–dgalactopyranoside (IPTG)-induced mazF expression cassette, in which the E. coli mazF was cloned right after the IPTG-inducible promoter Pspac and integrated inside the chromosome of B. subtilis TMO(Morimoto et al., 2009). Bacillus licheniformis BL-109 derived from B. licheniformis CBBD302 (Niu et al., 2009) by deleting the thermophilic -amylase coding gene amyL was employed because the host cell for enzyme overexpression. Bacillus licheniformis BS-109 was previously created in this laboratory by integrating one copy of amyS| Journal of Industrial Microbiology and Biotechnology, 2022, Vol.PMID:24182988 49, No.Fig. two The helper plasmid pUB-Tet curing as well as the expression plasmid pUB -EX1 integration in B. licheniformis. (a) The ratio of cells preserving helper plasmid after 4 times subculturing; error bars indicate normal deviation from three parallel experiments. (b) The evaluation of plasmid pUB-Tet and pUB -EX1 existence in B. licheniformis. The concentration of tetracycline in LB plates was 2 g/ml as well as the concentration of kanamycin inside the LB plate was 20 g/ml. Arrows indicate colonies with helper plasmid curing; the open circle marks the colony in which pUB -EX1 was integrated into the chromosome.Fig. three Functional verification of pUB-MazF and pUB -EX1 in B. subtilis and B. licheniformis. (a) The replication of pUB-MazF in B. subtilis WB600 and B. licheniformis BL-109. (b) The replication and integration of pUB -EX1 using the assistance of pUB-MazF in B. subtilis WB600 and B. licheniformis BL-109. Culture circumstances had been marked at the top rated of each and every section. For verification of plasmid replication and integration, the cultures had been spread on LB plates containing tetracycline (15 g/ml in B. subtilis or five g/ml in B. licheniformis) and/or 20 g/ml kanamycin.encoding the mature peptide of a thermoph.