Part:BBa_K3271027
HF Phusion Polymerase
The pSB1K3 backbone contains within its prefix and suffix the coding sequence of High-Fidelity Phusion Polymerase. The coding sequence, derived from Escherichia phage T5, is codon-optimized for expression in E. coli and for enhanced performance. HF Phusion Polymerase catalyzes DNA amplification 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 or for day-to-day PCR amplifications.
Design, Cloning, Expression, and Purification of HF Phusion Polymerase for the making of DIY Gibson Assembly Kit
Thermo Fisher Scientific websites were consulted to identify the patented amino acid sequence for the commercialized enzyme (HF Phusion Polymerase). 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 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 digestion of resultant plasmids (with NcoI and SalI in NEBuffer 3.1). Digests were gel electrophoresed to screen for successful inserts.
Plasmids which tested positive for the polymerase 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 was 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 polymerase 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 ensure overexpression of the desired protein. Remainder of the induced culture was pelleted, lysed, and nickel-column purified with imidazole washing step. Polymerase was stored in 50% glycerol at -20°C.
Testing the function of the purified polymerase
To ensure that the components of the Gibson Assembly Kit can function together, individual enzyme function had to be confirmed. A plethora of PCR reactions were performed using the Phusion polymerase. The home-made polymerase successfully amplified amplicons ranging in length from 200 bp to above 4000 bp at elongation temperatures ranging from 50°C to 75°C and at 30 cycles of heating to 98°C for primer annealing and cooling to the aforementioned elongation temperatures. This Phusion polymerase can amplify purified DNA and even genomic DNA with cellular debris from yeast and colony PCR.
NOTE: It is strictly forbidden to use the given sequence for profit, as this sequence has already been commercially patented.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1430
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1408
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