Part:BBa_K3271026

Taq Ligase in pSB1K3

The pSB1K3 backbone contains within its prefix and suffix the coding sequence of Taq ligase. The coding sequence, derived from Thermus aquaticus, is codon-optimized for expression in E. coli. Taq ligase forms a phosphodiester bond between 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA.Transfer the coding sequence into a high-copy, protein-expression backbone. Express the protein and purify it, using nickel column purification, for instance. The purified protein can thus be used in 'home-made' Gibson Assembly kits.

Design, Cloning, Expression, and Purification of Taq Ligase for the Making of DIY Gibson Assembly Kit

After cross-referencing several academic papers and journals, the amino acid sequences for Taq ligase on UniProt (B7A6G7) was identified. Using IDT codon optimization tool, amino acid sequence was converted into DNA sequences optimized for expression in E. coli. In silico, NcoI and SalI restriction sites were added to the 5’ and 3’ ends of the DNA sequence to each coding sequence. Primers were designed to amplify the entire DNA sequence. Both primers and DNA sequence were synthesized via IDT.

Synthon was amplified via PCR (Figure 1) and digested using NcoI + SalI in NEBuffer 3.1 and then ligated using T4 DNA ligase, into pHis-Parallel 2 high expression vector. Ligate was transformed into DH5α E. coli, plated on LB-AMP, and incubated overnight. Colonies were arbitrarily selected for screening via miniprerp plasmid extraction and PCR verification for the inserts. PCR Products were gel electrophoresed to screen for successful inserts (Figure 2).

Figure 1. The PCR of Taq Ligase synthon was difficult, due to partial annealing of the primers to other regions of the synthon. The desired band (the brightest band, at 2031 bp, corresponding to the desired Taq ligase amplicon) was the major product with some minor impurities.

Figure 2. Plasmids extracted from arbitrarily chosen colonies were digested with NcoI and SalI to identify plasmids containing the desired Taq Ligase insert. The 10th lane from the left contains a band corresponding to the plasmid backbone (top band) and another band corresponding to the Taq liges insert at about 2000 bp.

Plasmids which tested positive for the Taq ligase insert were transformed into a Rosetta E. coli strain and plated on LB with ampicillin and chloramphenicol. 10 colonies were amassed and transferred into a 300 mL LB growth medium consisting of both chloramphenicol and ampicillin and incubated overnight in a 200 RPM shaker at 30°C for the OD600 to attain a value between 0.4 and 0.6. A 5mL aliquot of the culture was transferred to a larger flask 1 L LB chloramphenicol and ampicillin. Diluted cultures were grown for 4 hours at 25°C, 200 RPM. 1.5mL uninduced sample stored for later comparison and the remaining culture in the flask was treated with 100uL of IPTG to induce expression of Taq ligase insert. Flasks were returned to the 25°C shaker, 200 RPM, and incubated for an additional 2 hours. 1.5mL of induced sample wad stored at -80°C freezer after flash freezing with liquid nitrogen. Induced and uninduced cultures were pelleted, lysed via bead beat in 250µl of 1X SB with PMSF, and run through a 15% polyacrylamide gel. Gel was stained using Coomassie stain to verify overexpression of the desired protein (Figure 3). Remainder of the induced culture is to be pelleted, lysed, and nickel-column purified with imidazole washing step. Taq ligase will be stored in 50% glycerol at -20°C.

Figure 3. The induced sample of Taq ligase culture shows a new protein right around 80 kDa, the appropriate molecular mass for Taq ligase.

Next step would be to quantitate the catalytic activity of the enzyme independently of the other enzymes found in the same Gibson Assembly Master Mix. Then, the efficiency of the Gibson Assembly Master Mix should be assessed by swapping one of the commercial Gibson Assembly enzymes, with one of our ‘home-made’ versions.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 92
  Illegal BamHI site found at 633
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1026
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 142
  Illegal SapI.rc site found at 292