Notebook

7/8 (online)

Use Salmonella Enterica Genome as PCR template, amplify fadB enzyme through PCR; use pUC57-ter as PCR template, amplify gene ter through PCR;
use pUC57-bktB as PCR template, amplify gene bktB through PCR;
use Escherichia coli genome as PCR template, amplify gene ydi1 through PCR
Gel extraction.
Double-enzyme digest pETDuet-1 with Nco1/BamH1, gel extraction.
Ligate fadB fragment and digested pETDuet-1 vector using one-step cloning method, transfer the recombinant plasmid into DH5α competent cells and coat on solid LB medium.
Double-enzyme digest pCDFDuet-1 using Nco1/BamH1, extract the gel.
Ligate bktB fragment and digested pCDFDuet-1 vector using one-step cloning method, transfer the recombinant plasmid into DH5α competent cells and coat on solid LB medium.

Inoculate monoclonal colonies of pETD-fadB and pCDFD-bktB into LB medium.
Use pETD-EGFP plasmid as PCR template, amplify GFP through PCR, and gel extraction. (F, R primers carry homologous recombination sequences)
Double-enzyme digest pET28a plasmid using Nco1/BamH1, extract the gel.
Mix the GFP fragments and the digested plasmid fragments at a molar concentration ratio of 3:1, and use Hieff homologous recombinase to ligate, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium.

Extract pETD-fadB and pCDFD-bktB plasmid, and verify whether they are successfully constructed through Nco1/BamH1 double-enzyme digest.
Inoculate the monoclonal colonies of pET28a-EGFP into LB medium.

Extract the pET28a-EGFP plasmid, and verify whether it is inserted into the original plasmid template using Nco1/BamH1 double-enzyme digest.
Double-enzyme digest pETD-fadB using NdeI/XhoI, gel extraction.
Ligate gene ter fragment with the digested pETD-fadB vector using the one-step cloning method, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium.
Double-enzyme digest pCDFD-bktB using NdeI/XhoI, gel extraction.
Ligate gene ydi1 fragment with digested pCDFD-bktB fragment using the one-step cloning method, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium.

Inoculate monoclonal colonies of pETD-fadB-ter and pCDFD-bktB-ydi1 into LB medium.

Extract pETD-fadB-ter and pCDFD-bktB-ydi1 plasmid, and verify whether they are inserted into the original plasmid template through NdeI/XhoI double-enzyme digest.
Use pET28a-EGFP plasmid as PCR template, and amplify 4 different kinds of T7 promotor mutant (C1~C4) through PCR.
Dpn1 digestion, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium.

Extract T7 point mutation plasmids, and verify whether the point mutation was successfully constructed using sanger sequencing.

Transfer the correct pET28a-EGFP_C1-C4 plasmids to BL21(DE3) competent cells, and coat on a solid LB medium.

Incubate BL21(DE3) strain containing pET28a-EGFP_C1-C4 plasmids in 5mL LB medium overnight.

1% transform the cultured medium into 25mL fresh LB medium until OD₆₀₀=0.5.
Induce protein expression using IPTG, and incubate for 3h.
Measure OD₆₀₀ and the fluorescence intensity of GFP to select the optimal mutation.
Use pETD-fadB-ter and pCDFD-bktB-ydi1 as PCR templates, amplify the optimal T7 point mutation plasmid using PCR.
Dpn1 digestion, transfer the recombinant plasmid into DH5α competent cells, and coat on a solid LB medium plate.

Inoculate the monoclonal colonies into LB medium.
Extract plasmids, and verify whether the point mutation was successfully constructed using sanger sequencing.
Transform the correct plasmids into BL21 (DE3), coat on a solid LB medium plate.

Inoculate the BL21 (DE3) monoclonal colonies into LB medium, and incubate at 37℃ overnight.
1% transform the cultured medium into 25mL fresh LB medium until OD₆₀₀=0.5.
Induce protein expression using 0.5 mM IPTG, and incubate for 40h.
SDS-PAGE gel electrophoresis verification.