Construction of Mutated E. coli K88ac LT(S63K)ΔSTb From {"type":"entrez-nucleotide","attrs":{"text":"C83902","term_id":"2706834","term_text":"C83902"}}C83902

Deletion of STb was performed using an E. coli gene knockout kit (Jiangsu RuiYang BioTech Co., Ltd., China) as protocol and the construction process as shown in Figure 1A. Briefly, PCR fragments using primers P1/P2 and P3/P4 amplified from the {"type":"entrez-nucleotide","attrs":{"text":"C83902","term_id":"2706834","term_text":"C83902"}}C83902 strain were cloned into restriction endonuclease sites EcoR I/Sma I and Sma I/Hind III of plasmid pUC18, then the dif-Gm-dif DNA digested with Sma I was inserted into the restriction endonuclease Sma I site. The constructed plasmid above was used as the template, and the recombinant cassette containing the left homologous arm, right homologous arm, and dif-Gm-dif amplified by PCR using primers P5/P6 was transformed into {"type":"entrez-nucleotide","attrs":{"text":"C83902","term_id":"2706834","term_text":"C83902"}}C83902 with plasmid pKD46 induced by L-arabinose at a final concentration of 4 μg/ml. After homologous recombination, the gentamicin resistance gene (Gm^r) was deleted by the dif/Xer recombinant system of the E. coli, and a 34-bp sized dif sequence was left in the large plasmid of the E. coli named K88ac ETECΔSTb, which was verified by PCR using primers P7/P8.

Construction of mutated E. coli K88ac LT(S63K)ΔSTb. (A). Deletion of STb to construct of E. coli K88ac ETECΔSTb by λ-Red homologous recombination; (B). Construction of E. coli K88ac LT(S63K)ΔSTb based on E. coli K88ac ETECΔSTb by mutating nucleotides 187–189 of LT from TCT (serine) to AAA (lysine) using λ-Red homologous recombination.

Using plasmid pSG76-CS as template, another recombinant cassette containing left and right homologous arms, mutated nucleotides, and chloramphenicol resistance gene (Cm^r) with two endonuclease I-SceI sites on two sides was amplified by overlapping PCR using primers P9/P10/P11. The PCR product was transformed into K88ac ETECΔST1 with plasmid pKD46 induced by L-arabinose to insert the amplicon into LT by λ-Red recombination. Then, plasmid pKD46 was removed at a temperature of 40°C and the plasmid pSTKST was transformed into the recombinant E. coli. After being induced by chlorotetracycline (Shanghai Yuanmu BioTech Co., Ltd., China) at a final concentration of 30 μg/ml, endonuclease I-SceI expressed by plasmid pSTKST was used to delete the Cm^r gene. The large plasmid in E. coli was repaired by the method of broken-end recombination, and the mutation was fixed in LT. Then, plasmid pSTKST was removed at 40°C and the recombinant E. coli named K88ac LT(S63K)ΔSTb was constructed and verified by sequencing the PCR product amplified using primers P12/P13 as Figure 1B.