Part:BBa_K3271025
T5 Exonuclease in pSB1K3
The pSB1K3 backbone contains within its prefix and suffix the coding sequence of T5 exonuclease. The coding sequence, derived from T5 phage, is codon-optimized for expression in E. coli. T5 exonuclease cleaves linear or nicked double-stranded DNA in the 5' to 3' direction. 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 T5 exonuclease for the making of DIY Gibson Assembly kit
After cross-referencing several academic papers and journals, the amino acid sequences for T5 exonuclease on UniProt (Q9H816) was identified. Using IDT codon optimization tool, amino acid sequence was converted into DNA sequences optimized for 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 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).
Plasmids which tested positive for the T5 exonuclease 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 T5 exonuclease 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 (Figure 3). 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 4). Remainder of the induced culture is to be pelleted, lysed, and nickel-column purified with imidazole washing step. T5 exonuclease will be stored in 50% glycerol at -20°C.
Although we did not have the chance quantitate the catalytic activity of T5 exonuclease, our pellet indicates that the enzyme has likely retained its function, as its over-expression could have resulted in the appreciable decline in cell harvest due to its toxic effects on the host.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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