Overview

Lambert iGEM utilized padlock probes with rolling circle amplification (RCA) see RCA: Overview to detect various microRNAs (miRNAs) that are upregulated in correlation with coronary artery disease (CAD) (Fichtlscherer et al., 2010). The RCA reaction utilizes phi29 DNA polymerase to amplify the padlock probe sequence (Jonstrup et al., 2006 & Fang et al., 2021). However, a single RCA reaction is $8.98, with phi29 DNA polymerase making up 56% of the cost. Moreover, multiple reactions need to be performed for a more thorough diagnosis, restricting its use to privileged communities, and making it unaffordable for most demographics. To increase the preventative aspect of healthcare and decrease the cost of the overall assay, we produced phi29 DNA polymerase through protein purification.

Protein Purification

Protein purification is commonly used to isolate proteins from a complex mixture (Labrou, 2014). Specifically, immobilized metal affinity chromatography (IMAC) is a protein purification technique used to purify recombinant proteins fused to six consecutive histidine tags (his-tags). His-tags are short DNA sequences inserted at an end terminal of the DNA encoding protein in a recombinant plasmid (see Fig. 1).

Figure 1. Recombinant plasmid containing DNA encoding protein fused with His-tags.

To purify phi29 DNA polymerase, we utilized an existing plasmid from Addgene that produces the protein attached to six His-tags (see Fig. 1). We isolated the plasmid inside DH5α through miniprep then transformed the plasmid into BL21 for protein expression and cultured it in Luria Broth (LB) growth medium. The resulting cells in the solution were then lysed and filtered through a column that contained positively-charged nickel affinity resin, prompting a bond with the negatively-charged His-tags and polymerase (see Fig. 2) (Corbin & Falke, 2013). To remove the other parts of the lysate and elute the proteins from the columns, we added the His-Wash and His-Elution buffers, respectively (see Fig. 2).

Figure 2. phi29 DNA purification through the use of nickel column and the chelation bond of the His-tag and nickel affinity resin.

We then performed a Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique commonly used for protein separation to measure the protein’s size to ensure only the correct protein has been purified.

Design

To produce phi29 DNA polymerase, Lambert iGEM initially used TAS Taipei part, BBa_K3352009 (see Fig. 3), with pet-11a-ABLE (see Fig. 4), an ampicillin-resistant and IPTG-inducible 6xHis-TEV-ABLE backbone from Addgene (Addgene: pet11a-ABLE, 2023). However, when we attempted to purify the pet-11a-ABLE backbone, we obtained repetitive impure results. Therefore, our team acquired pGS-21a his6-GST-WTPhi29DNAP, a plasmid that produces an affinity-tagged phi29 DNA polymerase (see Fig. 5), allowing us to bypass restriction digest and purify the protein (PGS-21A His6-GST-WTPHI29DNAP (plasmid #166863)).

Figure 3. Construct of pET T7 Promoter with Modified UTR, Extended RBS, phi29 DNA polymerase, and T7 Terminator (BBa_K3352009)

Figure 4. Full Sequence Map of pet11a-ABLE from SnapGene

Figure 5. Full Sequence Map of pGS-21a his6-GST-WTPhi29DNAP from SnapGene

Results

After running our purified phi29 DNA polymerase from immobilized metal affinity chromatography (IMAC) on the Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)see Experiments: Protein Purification, Lambert iGEM saw bands around 50kD ~ 75kD. When we compared this band to the commercial phi29 DNA polymerase from New England Biolabs, it indicated that the protein was purified as the bands appeared in the same area (see Fig. 6).

Figure 6. SDS-PAGE with purified phi29 DNA polymerase, displaying bands at 50kD ~ 75kD

Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA) by comparing the rolling circle products (RCP) of the commercial and purified phi29 DNA polymerase. We added the same volume of both to RCA reactions and ran the rolling circle products on a gel. We saw fluorescent bands of DNA very close to the wells, indicating that both the RCA assay utilizing commercial and purified enzyme reactions were successful (see Fig. 7).

If we buy phi29 DNA polymerase from New England Biolabs, it will cost $251.00 for 1,250 units. However, after we utilized the NEBExpress Ni Spin Columns ($10 per prep to purify the enzyme), we were able to produce 200ul in one prep, or approximately 2,000 units. Because, it can be assumed that the unit between the commercially bought enzyme and our purified enzyme is similar, if commercially bought, this 200ul amount would cost around $400. Therefore, we can determine that our purified protein is significantly more affordable than the commercially bought protein.

Figure 7. Gel comparing the rolling circle product (RCP) between the team’s purified phi29 DNA polymerase and that commercially available phi29

In the future, we plan to more accurately quantify the activity of the purified units of phi29 DNA polymerase. Additionally, we hope to test the accuracy of our enzyme by quantifying the RCP using linear DNA probes (see RCA: outputs) to validate its use of point-of-care testing.

References