Clonexpress 2 one step cloning kit c112 01

The ΔycgF strain was obtained from the E. coli W1688 strain by knocking out the target gene ycgF using the CRISPR/Cas9 gene-editing technology. The primers used are shown in Table S1 in the supplemental material. The specific operation is as follows. The donor DNA fragment and pTarget plasmid containing the N₂₀ sequence were constructed and introduced into competent E. coli W1688 cells containing the pCas plasmid. Using Kan (50 mg/L) and Str (40 mg/L) as resistance markers, the transformed cells were screened to select the cells lacking ycgF.
To express nMagHigh and pMagHigh on the surface of E. coli, we fused these proteins to the N terminus of OmpX (45 (link)). To insert the nMagHigh gene into the plasmid pET28a, the gene cassettes nMagHigh-linker-OmpX and pMagHigh-linker-OmpX were first synthesized by Tsingke Biotechnology Co., Ltd. Then, using the ClonExpress II one-step cloning kit C112-01 (Vazyme Biotech Co., Ltd.), these genes were inserted into the BglII restriction sites of pET28a-EGFP and pET28a-mCherry, respectively. Thus, plasmids pET28a-EGFP-nMagHigh-OmpX and pET28a-mCherry-pMagHigh-OmpX were obtained. The constructed plasmid was transformed into ΔycgF using the heat shock transformation method, and the transformants were selected using Kan (50 mg/L).

Escherichia coli W1688 (CCTCC M2015233) was an L-threonine producer obtained from E. coli MG1655 (ATCC47076) by mutation and molecular modification. It could not produce biofilms apparently. All strains and plasmids used in this work are listed in Tables 1, 2, respectively. The fimH gene was amplified from the genomic DNA of E. coli W1688. The fimH gene and plasmid pET28a (with restriction enzyme XbaI and NcoI) were ligated by using the ClonExpress II One Step Cloning Kit C112-01 (Vazyme, Nanjing, China), resulting in a plasmid pET28a-fimH. The final engineered strain was named E. coli W1688-fimH^* with Kanamycin resistance for screening. On the other hand, fimH from E. coli W1688 was deleted by Red homologous recombination, resulting in an E. coli W1688-ΔfimH (Madyagol et al., 2011 (link)). Briefly, a PCR-generated Kanamycin resistance marker was used as knock-in DNA fragment. The Kanamycin resistance marker consisted of a Kanamycin resistance sequence in plasmid pKD4 and homologous regions (50–100 bp) flanking the target locus. The knock-in component was transformed into strain E. coli W1688-pKD46 using Bio-Rad electroporation system set at 2.0 kV, 25 mF with a 200 Ohm pulse controller.

Escherichia coli MG1655 and BL21(DE3) were used in this study.
All strains and plasmids used in this work are listed in Table 1. Primers used in this study are listed in Supplementary Table 1. Vector pET30a-hEGF was used to express hEGF in BL21(DE3) and its modified strains. The biofilm genes bcsB, fimH, and csgAcsgB were amplified from the genomic DNA of E. coli MG1655. These genes were individually ligated to the expression plasmid pBbE1a (with restriction enzyme BglII and AvrII) by using the ClonExpress II One Step Cloning Kit C112-01 (Vazyme, Nanjing, China) and then were expressed in MG1655 and BL21(DE3), respectively, resulting in plasmid-based expression strains MG1655-bcsB⁺, MG1655-csgAcsgB⁺, MG1655-fimH⁺, BL21-bcsB⁺, BL21-csgAcsgB⁺, and BL21-fimH⁺, respectively. For genome-integrated expression, the bcsB, fimH, and csgAcsgB were inserted in the moaE site by CRISPR, under the trc promoter, resulting in genome-integrated expression strains MG1655-bcsB*, MG1655 -csgAcsgB*, MG1655-fimH*, BL21-bcsB*, BL21-csgAcsgB*, and BL21-fimH*. The genes moaE, gshB, yceA, and ychJ in E. coli MG1655 and BL21(DE3) were individually deleted by CRISPR, using pCas and pTarget (Jiang et al., 2015 (link)), resulting in MG1655 ΔmoaE, MG1655 ΔgshB, MG1655 ΔyceA, MG1655 ΔychJ, BL21 ΔmoaE, BL21 ΔgshB, BL21 ΔyceA, and BL21 ΔychJ.