@@ -37,13 +37,19 @@ To produce phi29 DNA polymerase, Lambert iGEM initially used TAS Taipei part, BB
## 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](https://2023.igem.wiki/lambert-ga/experiments/), 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 most likely purified as the bands appeared in the same area (see Fig. 6).
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](https://2023.igem.wiki/lambert-ga/experiments/), 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).
caption='Figure 6. SDS-PAGE with purified phi29 DNA polymerase, displaying bands at 50kD ~ 75kD'/>
However, due to the oversaturation of the Coomassie G-250 Stain and shortage of time, we were unable to see a clear band on the SDS-PAGE. Furthermore, we were not able to compare the cost of our purified protein with our commercially bought protein as each prep of purification has to be compared individually due to unit differences. In the future, Lambert iGEM hopes to purify phi29 DNA polymerase to achieve a clear band confirming the enzyme, compare the cost of our purified protein to commercially bought protein, and finally, test its efficacy through the rolling circle amplification (RCA) assay further demonstrating its affordability.
Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA). We ran the rolling circle products on a gel and saw fluorescent bands of DNA very close to the well, indicating that the RCA reaction was successful (see Fig. 7).
caption='Figure 7. Gel comparing the rolling circle product (RCP) between the team’s purified phi29 DNA polymerase and that commercially available phi29'/>
However, we were not able to compare the cost of our purified protein with our commercially bought protein as each prep of purification has to be compared individually due to unit differences. In the future, Lambert iGEM hopes to purify phi29 DNA polymerase and compare the cost of our purified protein to commercially bought protein.
## References
<Reference> Falke, J. J., & Corbin, J. A. (2013). Affinity Tags for Protein Purification. Encyclopedia of Biological Chemistry. Retrieved June 10, 2023, from https://doi.org/10.1016/B978-0-12-378630-2.00173-0 </Reference>
