構建整合T7啟動子的Nissle 1917過程：Genomic DNA from E. coli BL21(DE3) was used as a template to obtain the T7-RNA polymerase gene. All oli gonucleotides used in the construction are listed in Addi tional file 1: Table S3. Via two PCR reactions overlapping primers (lac-op-fwd, lacUV5-HindIII-fwd) were used to add the lac operator and lacUV5 promoter to the 5′-end of the T7 RNA polymerase gene to allow for isopropyl β-d-thiogalactopyranoside (IPTG) inducible gene expres sion. A kanamycin resistance cassette, with flanking FRT sites, was amplified from pKD13 and ligated via a SalI restriction site to the 3′-end of the T7-RNA polymerase gene in order to generate a selection marker for the fol lowing homologous recombination reaction. The thus obtained lacUV5-T7-FRT-kan-FRT (T7/Kan) fragment was purified via gel extraction and blunt end cloned into the plasmid pYP168 [17] via a SmaI restriction site. This product (pUC-T7-FRT-kan) was then used as a PCR tem plate to add 50 bp of homologous sequences from the malEFG operon of E. coli Nissle 1917 (T7-mal-fwd and T7-mal-rev) to the expression cassette at both ends. The fragment was purified via agarose gel extraction.

參考文獻：https://doi.org/10.1186/s12934?020?01447?5