Construction of an E. coli strain (W3110) harboring chromosomal 3xFLAG-tagged RutR

Title: Construction of E. coli W3110 Strain with Chromosomal 3xFLAG-Tagged RutR

Authors: Shun Tu, Sheng-ce Tao

Affiliation: Institute of systems biomedicine, Shanghai Jiao Tong University, China, 200240

Background: The protein YcgC has been identified to possess deacetylase activity towards the substrate protein RutR. To investigate whether YcgC can deacetylate RutR in vivo, we devised an experiment utilizing the highly efficient homologous recombination capabilities of E. coli, facilitated by the Red recombination system. Given the lack of high-quality antibodies for many E. coli proteins, we opted to append a 3xFLAG tag to the C-terminus of the chromosomal RutR gene. This tag serves to facilitate the immunoprecipitation and subsequent detection of RutR from the total protein lysate.

1. Materials:

• Anti-FLAG mouse monoclonal antibody (F1804), Anti-FLAG rabbit polyclonal antibody (F7425) from Sigma.
• Secondary antibody (926-32213 IRDye 800CW) from LI-COR Biosciences.
• E. coli AG1 from Agilent (Cat. 200274).
• E. coli W3110 strain provided by Dr. Jiaoyu Deng, Wuhan Institute of Virology, Chinese Academy of Sciences.
• 3xFLAG sequence synthesized by GenScript Biotech Co.
• KOD-FX PCR mix (TOYOBO, A111-01).
• 1X TBST for antibody dilution.

2. Methods:

2.1 PCR Amplification and Processing of the Target Gene:

• Amplify the full-length RutR_3X FLAG fragment using pUC57 plasmid as a template with designed primers.
• Primer sequences:
  • Upstream: 5’-GCTGTTAATGGATGAACTGACGG-3’
  • Downstream: 5’-GATGCGGTGATCTTCCACGGTG-3’
• PCR reaction setup (50 μL):
  • 2X KOD Polymerase Buffer 25 μL
  • dNTP (2.5 mM) 1 μL
  • Primer sense (20 μM) 1 μL
  • Primer anti-sense (20 μM) 1 μL
  • pUC57 plasmid 0.5 μL
  • KOD-FX DNA Polymerase 1 μL
  • ddH2O to final volume
• Cycling conditions: 94 °C, 5 min; (94 °C, 1 min; 55 °C, 40 sec; 72 °C, 2 min) x 30 cycles; 72 °C, 10 min.
• Verify PCR product on 1% agarose gel, purify using a DNA Gel Recovery Kit (TIANGEN, Beiijng, China) and quantify with NanoDrop (Thermo Fisher Scientific, Waltham, MA).
• Treat with DpnI enzyme at 37 °C for 2 hrs to digest methylated DNA and recover the linear fragment.

2.2 Design and Synthesis of Full Gene Sequence of RutR with C-terminal 3X FLAG Tag:

• Synthesize a 2,079 bp sequence containing RutR (according to E. coli K12 strain) C-terminal 265 bp, 3X FLAG tag (66 bp), stop codon, KANA resistance gene (1,476 bp), and RutR downstream flanking sequence (147 bp).
• The synthesized sequence is cloned into pUC57 plasmid and named RutR_3X FLAG, then sequenced for verification.

2.3 Knock-in of the Synthesized Target Gene into the Host Strain:

2.3.1 Preparation of Routine Competent Cells:

• Inoculate E. coli W3110 in 5 mL LB medium and incubate overnight at 37 °C.
• Grow single colonies on LB agar at 37 °C.
• Inoculate a single colony into 3 mL LB and incubate overnight at 37 °C.
• Dilute 1 mL of overnight culture into 100 mL LB and incubate until OD₆₀₀ ~ 0.5.
• Chill on ice for 10 min, centrifuge at 4 °C, 4000 rpm, and wash with ice-cold 0.1 M CaCl₂.
• Resuspend in 2 mL ice-cold CaCl₂, aliquot 100 μL per tube, and store at -80 °C.

2.3.2 Preparation of Competent Cells with Red Recombination Function:

• Transform pKD46 plasmid (the Red system) into W3110 competent cells and incubate with ampicillin at 30 °C.
• Add L-arabinose to induce Red recombination and incubate for 1.5 hrs.
• Centrifuge, wash with sterile water and 10% glycerol, and resuspend in 10% glycerol.
• Aliquot 80 μL per tube and store as described.

2.3.3 Electroporation and Verification of RutR_3X FLAG Knock-in:

• Electroporate DpnI-treated RutR_3X FLAG fragment into pKD46-transformed W3110 cells using a MicroPulser Electroporator from Bio-Rad.
• Select on LB agar with kanamycin and verify colonies by PCR using Kanamycin gene fragment primers.

2.4 Validation of Knock-in Using 3xFLAG Antibody:

• Enrich RutR_3X FLAG protein from total bacterial lysate using anti-FLAG antibody and Protein A/G beads (GE, Boston, MA).
• Perform Western blot analysis to detect the presence of RutR_3X FLAG protein.

3. Validation of Knock-in:

3.1 Enrichment of RutR_3X FLAG Protein:

• Thaw bacterial cells, lyse in pre-cooled lysis buffer, and clarify by centrifugation.
• Bind target protein using anti-FLAG antibody and collect with Protein A/G beads (GE, Boston, MA).

3.2 Western Blot Analysis:

• Resolve proteins on SDS-PAGE, transfer to a membrane, and block.
• Probe with anti-acetylation antibody, followed by secondary antibody detection.
• Analyze the results using an Odyssey scanner (Li-Cor Biosciences).

References:

1. Poteete AR, "What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function." FEMS Microbiol Lett, 2001. 201(1): 9-14.
2. Tu S, et al. "YcgC represents a new protein deacetylase family in prokaryotes." eLife, 2015; 4: e05322.