Team:Wisconsin-Madison/

Expression and Purification of Enzymes

Gibson Assembly

Scientists have long possessed the knowledge to synthesize natural and synthetic DNA sequences by combining two or more pieces of DNA. Known as recombinant DNA technology, these methods became widely used upon the discovery of endonucleases and DNA ligases. Over the years, methods have become increasingly efficient, leading to many new research discoveries. In 2009, Daniel Gibson published a paper outlining his efficient new method to combine and clone large pieces of DNA. Additionally, the method provides greater selectivity than previous cloning methods involving restriction enzymes. The method described by Gibson involves the use of commercially available enzymes, namely Taq DNA ligase (New England Biolabs, NEB), Phusion DNA polymerase (NEB), and T5 exonuclease (Epicentre).

The Gibson Assembly utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. The reaction proceeds as shown in figure 1: First, the exonuclease chews back the 5' ends of the overlapping pieces of DNA, allowing them to anneal. The amount of exonuclease must be finely tuned so that long enough sticky ends are created for the overlaps to anneal before the exonuclease becomes heat inactivated, but not a large enough amount of exonuclease that the DNA is destroyed. The polymerase then fills in the gaps created by the exonuclease until the ligase can join the strand, creating a single, unscarred, double-stranded piece.

Figure 1. Gibson, D.G. et al. Nat. Methods 343-345 (2009)

The Gibson Assembly is extremely useful for cloning. As the figure below shows, a gene can be designed to overlap a backbone plasmid. PCR can then be used to create the overlapping insert and backbone pieces, which can then be Gibson assembled.

This groundbreaking new method has proven useful in many labs, but the expense of the necessary enzymes may be costly for smaller research labs and universities. To overcome this problem, we synthesized and cloned Pfu polymerase, Taq Ligase, and T5 exonuclease, essentially creating our own in-house enzyme mixture. A lawyer at the UW-Madison Law & Entrepreneurship Clinic was contacted regarding the legality of synthesizing these patented enzymes. It was determined that we were not infringing on the patent, as the project’s only motivation was to satisfy our idle curiosity, and is not intended bring our lab group financial gain or significant notoriety. We hope this project will be a helpful educational tool for introductory biology students, while also producing something that is useful and meaningful.

Purification of Enzymes

Following the completion of purification, a number of tests were carried out to verify the legitimacy of the protocol. Firstly, the eluted protein fraction from the Ni-NTA columns were tested against an uninduced control to ensure that a protein of interest had in fact been expressed and purified. This was done by SDS-PAGE electrophoresis. These results for taq ligase are shown in figure 2 and the results for T5 exonuclease are shown in figure 3, below.

Figure 2. This gel shows elution fractions from the taq ligase purification. It can be seen that taq ligase is expressed in very large amounts in the fractions from induced cells(extremely dark band). It can also be seen that the elution is significantly more pure than the unpurified fractons. Although a smaller band of taq ligase can be seen in samples from the uninduced fractions, the expression in the uninduced cells can be attributed to leakyness in the T7 expression system.

Figure 3. This gel shows elution fractions from the T5 exonuclease purification. It can be seen that T5 is expressed in very large amounts in the fractions from induced cells(extremely dark band). It can also be seen that the eluted fraction has significantly fewer impurities than the unpurified fractons. Although a smaller band of T5 Exonuclease can be seen in samples from the uninduced fractions, the expression in the uninduced cells can be attributed to leakyness in the T7 expression system.

Once the expression had been verified, the protein concentration of the purified enzyme was found using a bradford assay. We found that we produced 3 mL of each enzyme, with the concentration of taq ligase around 2.274 mg/mL and the concentration of T5 exonuclease produced around 2.600 mg/mL

Testing the Efficacy of Enzymes

T5 Exonuclease

T5 exonuclease tends to be a highly effective enzyme, and therefore an assay was used to test the efficacy of our enzyme against the commercially sold T5 exonuclease (Epicentre). To prove that T5 exonuclease did not chew circular DNA, 1 uL various dilutions of T5 exonuclease were added to 5 uL of DH5α p102cherry plasmid DNA in Buffer 4 (New England Biolabs). The dilutions were incubated in a 37°C water bath for 30 minutes, followed by addition of 6x loading dye. Following incubation, the dilutions were added to an agarose gel and allowed to run. The gel was stained in ethidium bromide staining solution for 15 minutes with gentle agitation, followed by a 15 minute water wash.

Following this confirmation, T5 exonuclease was tested against commercially available T5 exonuclease on linear strands of DNA, with the goal of creating a dilution that matched the commercial T5 exonuclease when run out on a gel. 1 uL of various dilutions of T5 exonuclease were added to 1.3 uL of linear ds DNA and incubated at 37°C for 15 minutes. Following incubation, 6X loading dye was added to the dilutions and run out on an agaraose gel. The gel was stained with ethidium bromide solution and rinsed with water.

Taq Ligase

Taq ligase was used to . Upon discovering that Taq ligase cannot anneal 4-5 base pair overhangs created by restriction digest enzymes, a new method to test the enzyme performance was developed. λ DNA-Mono Cut Mix (New England Biolabs) containing a range of DNA lengths was used to select the appropropriate enzyme dilution. The ligase worked to conjoin the pieces of DNA, effectively creating larger pieces. 1 uL of selected Taq ligase dilutions were added to 2 uL λ DNA. The various dilutions were incubated in a thermocycler at 45°C for 15 minutes, followed by addition of 10 uL stop dye. Sequentially, the dilutions were heated to 70°C in a thermocycler for 10 minutes, and loaded onto a 0.7% agarose gel. The gel was stained with Ethidium bromide solution for 15 minutes, and washed with DI water.

PFU DNA Polymerase

PFU Polymerase has been successfully purified, and performance assays are in progress.

In Conclusion

T5 exonuclease, Taq Ligase, and pfu polymerase were effectively synthesized, purified with minor impurities, and determined to be effective in terms of use in Gibson Assembly. Both Taq Ligase (4.55 ug/mL) and T5 exonuclease (5.20 ug/mL) were diluted 500-fold due to being heavily concentrated. Pfu polymerase was concentrated as well, and diluted to a concentration of 1 mg/mL. All enzymes were stored at -20°C until use. A test-run of Gibson Assembly using our purified proteins showed comparable results to the commercially sold Gibson Master Mix based on amount of product formed.