Team:Athens/Contribution
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Contribution
Overview
Our team’s contribution can be divided into three distinct segments. Firstly, we contributed to the iGEM part library by adding information from literature to three part pages (BBa_K4103011, BBa_K1550007 and BBa_K4103013). Next, we added nine new parts in the iGEM registry with the necessary well-detailed information and lastly, we created a guide for safer handling of gblocks and genes from IDT and Twist Bioscience. We hope that these contributions will help future iGEM teams to work with these parts and guidelines to achieve their projects and make their iGEM journey easier.
Literature information about the DNA Polymerase Pfu
The DNA polymerase Pfu (Pfu polymerase), can be found in Pyrococcus furiosus, which is an extremophilic species of Archaea and it is a vital protein that is responsible for adding new nucleotides during DNA replication [1, 2]. DNA polymerase consists of 775 amino acids and has a molecular weight of 92 kDa. It has been found that it has a homologous structure to the α-like DNA polymerases of humans and to the DNA polymerase II, which is found in E. coli [3]. Due to its thermotolerans it has been widely used for Nucleic acid amplification through polymerase chain reaction (PCR) [4].
The main advantage of this enzyme is that it possesses 3´→5´ exonuclease activity (“proof-reading”), which allows it to reverse its direction and correct mismatched bases [1]. Thus, its fidelity is much better (1.3 x 10-6 mutation frequency/bp/duplication) than other thermotolerant DNA polymerases like Taq (8 x 10-6 mutation frequency/bp/duplication) [5].
Since error rate is important to be minimum in a number of techniques, there are several attempts to improve Pfu polymerase in that aspect with the most important being the Phusion variant [6]. Additionally, the Pfu can be attached to Sso7d which is a 7 kDa protein originating from Sulfolobus solfataricus, a hyperthermophilic archaebacterium [7]. Fusion to Sso7d has been proven to increase processivity, enabling longer amplifications and greater amplification speed [8].
Since Pfu is an important enzyme worldwide there have been reported many attempts to produce it and purify it. Originally Pfu polymerase was isolated directly from Pyrococcus furiosus, but growing this species is a challenge especially in large quantities [9]. Thus, it has been successfully attempted to express that enzyme in E. coli BL21 [10]. The purification of the protein has been done with many different ways like His-tag purification and with the use of weak cation exchange resins [11, 12]. Recently, a new simple method utilizing the tolerance to heat of the enzyme has been successfully used introducing a new level of simplicity for isolation and purification of thermotolerant proteins [13].
New Registry Parts
As part of our project we added ten new parts to the registry, writing information from literature whenever necessary. We hope that future teams can utilize these parts for the completion and enhancement of their own project.
| Part Name | Short Description | Part Type | Length (bp) |
|---|---|---|---|
| BBa_K4034002 | nrdA | Coding | 2286 |
| BBa_K4034003 | nrdB | Coding | 1131 |
| BBa_K4034004 | TSase | Coding | 795 |
| Part Name | Short Description | Part Type | Length (bp) |
| BBa_K4034006 | Pfu with His-tag | Coding | 2343 |
| BBa_K4034005 | RNR | Transcriptional unit | 3665 |
| BBa_K4034008 | pPfuT7 | Plasmid | 4697 |
| BBa_K4034009 | pAdAPTED | Plasmid | 6933 |
| BBa_K4034010 | pPfusionT7 | Plasmid | 4984 |
| BBa_K4034011 | pAdAPTED2T7 | Plasmid | 7319 |
Tips & Tricks for BioBricks Handling
Below you can find a preview of our guide on BioBricks handling or you can download it in pdf form here.
1. Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A., & Mathur, E. J. (1991). High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene, 108(1), 1–6. https://doi.org/10.1016/0378-1119(91)90480-y
2. Zheng, W., Wang, Q., & Bi, Q. (2016). Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli. The Protein Journal, 35(2), 145–153. https://doi.org/10.1007/s10930-016-9651-4
3. Uemori, T., Ishino, Y., Toh, H., Asada, K., & Kato, I. (1993). Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. Nucleic Acids Research, 21(2), 259–265. https://doi.org/10.1093/nar/21.2.259
4. Pavlov, A. R., Pavlova, N. V., Kozyavkin, S. A., & Slesarev, A. I. (2004). Recent developments in the optimization of thermostable DNA polymerases for efficient applications. Trends in biotechnology, 22(5), 253–260. https://doi.org/10.1016/j.tibtech.2004.02.011
5. Cline, J., Braman, J. C., & Hogrefe, H. H. (1996). PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic acids research, 24(18), 3546–3551. https://doi.org/10.1093/nar/24.18.3546
6. McInerney, P., Adams, P., & Hadi, M. Z. (2014). Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. Molecular Biology International, 2014, 1–8. https://doi.org/10.1155/2014/287430
7. Agback, P., Baumann, H., Knapp, S., Ladenstein, R., & Härd, T. (1998). Architecture of nonspecific protein–DNA interactions in the Sso7d–DNA complex. Nature Structural Biology, 5(7), 579–584. https://doi.org/10.1038/836
8. Wang, Y. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research, 32(3), 1197–1207. https://doi.org/10.1093/nar/gkh271
9. Fiala, G., & Stetter, K. O. (1986). Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 °C. Archives of Microbiology, 145(1), 56–61. https://doi.org/10.1007/bf00413027
10. Lu, C., & Erickson, H. P. (1997). Expression in Escherichia coli of the Thermostable DNA Polymerase from Pyrococcus furiosus. Protein Expression and Purification, 11(2), 179–184. https://doi.org/10.1006/prep.1997.0775
11. Dąbrowski, S., & Kur, J. (1998). Cloning and Expression in Escherichia coli of the Recombinant His-Tagged DNA Polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein Expression and Purification, 14(1), 131–138. https://doi.org/10.1006/prep.1998.0945
12. Sun, Z., & Cai, J. (2006). Purification of recombinant Pfu DNA polymerase using a new JK110 resin. Korean Journal of Chemical Engineering, 23(4), 607–609. https://doi.org/10.1007/bf02706802
13. Sankar, P. S., Citartan, M., Siti, A. A., Skryabin, B. V., Rozhdestvensky, T. S., Khor, G. H., & Tang, T. H. (2019). A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification. Iranian journal of microbiology, 11(2), 181–186.