• International Journal of Technology (IJTech)
  • Vol 11, No 5 (2020)

Cloning of DNA Polymerase I Geobacillus thermoleovorans SGAir0734 from a Batu Kuwung Hot Spring in Escherichia coli

Cloning of DNA Polymerase I Geobacillus thermoleovorans SGAir0734 from a Batu Kuwung Hot Spring in Escherichia coli

Title: Cloning of DNA Polymerase I Geobacillus thermoleovorans SGAir0734 from a Batu Kuwung Hot Spring in Escherichia coli
Kenny Lischer, Kevin Priyono Tansil, Mikael Januardi Ginting, Muhamad Sahlan, Anondho Wijanarko, Masafumi Yohda

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Cite this article as:
Lischer, K., Tansil, K.P., Ginting, M.J., Sahlan, M., Wijanarko, A., Yohda, M., 2020. Cloning of DNA Polymerase I Geobacillus thermoleovorans SGAir0734 from a Batu Kuwung Hot Spring in Escherichia coli. International Journal of Technology. Volume 11(5), pp. 921-930

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Kenny Lischer Universitas Indonesia
Kevin Priyono Tansil Universitas Indonesia
Mikael Januardi Ginting Universitas Indonesia
Muhamad Sahlan Universitas Indonesia
Anondho Wijanarko Universitas Indonesia
Masafumi Yohda Tokyo University of Agriculture and Technology
Email to Corresponding Author

Abstract
Cloning of DNA Polymerase I Geobacillus thermoleovorans SGAir0734 from a Batu Kuwung Hot Spring in Escherichia coli

Access to biological engineering has become a critical point of modern science development through polymerase chain reaction (PCR). One of the main components in this process is DNA polymerase, which copies the main template DNA. However, there is a lack of studies on the production of DNA polymerase from indigenous thermophilic bacteria in Indonesia. To examine this process, DNA polymerase I gene (DNA pol I) from Geobacillus thermoleovorans (isolated from Batu Kuwung, Banten, Indonesia) was transformed into Escherichia coli. The gene was cloned by the cut and ligation method using NcoI and BamHI restriction enzymes, which were ligated with a pET23d vector. The recombinant gene was overexpressed in E. coli  and identified by using SDS-PAGE of 10% acrylamide gel, showing that the protein molecular weight was approximately . This study successfully amplified the gene of interest, indicated by a high local similarity between the sequencing results and theoretical gene and positive intercellular protein expression. The results confirm that the study successfully cloned and synthesized recombinant DNA pol I of Geobacillus thermoleovorans from Batu Kuwung, Serang, Banten.

DNA pol I; DNA polymerase; Escherichia coli; Geobacillus thermoleovorans SGAir0734; Thermophilic bacteria

Introduction

Polymerase chain reaction (PCR) is a widely known method to amplify a DNA strain and its complement. The PCR products are critical for biological engineering developments and breakthroughs. Many things can be achieved through PCR, including gene amplification and detection, disease detection, and the creation of genetically modified organisms (GMO) (Beyer et al., 2002). One of the components required for PCR reaction is DNA polymerase, also known as the main enzyme (Valones et al., 2009). Without DNA polymerase, the extension process as the main stage of PCR will not occur (Maddocks and Jenkins, 2017). In Indonesia, despite having access to PCR technology, almost all DNA polymerase is still imported. 

DNA polymerase in both current global and local markets mostly derives from thermophilic bacteria  due to its higher resistance to denaturation and aggregation at high temperatures during conventional PCR (Maddocks and Jenkins, 2017; Gomes and Faísca, 2019). There are many papers regarding the finding that one of the first revolutionary thermophilic DNA polymerases comes from Thermus aquaticus (Engelke et al., 1990). There also other thermophilic bacterial-origin DNA polymerases such as Pyrococcus furiosus (Zheng et al., 2016), Bacillus stearothermophilus (Aliotta et al., 1996), or Geobacillus thermoleovorans species but from a different strain of ATCC 12980 (Li et al., 2017).

Thermophilic bacteria can be found in hot springs. Indonesia as a country is surrounded by the Pacific Ring of Fire and has many hot springs across the country. Therefore, the bioprospecting potential of indigenous thermophilic bacteria is very high. Although it seems promising, exploration of DNA polymerase from indigenous thermophilic bacteria in Indonesia remains low as there are still many thermophilic bacteria habitats that have no biodiversity studies published yet even the sites are considered public venues. So far, DNA polymerase I gene (DNA pol I) from Geobacillus thermoleovorans (from South Bandung hot springs) has been identified (Akhmaloka et al., 2008). There are still many unexplored thermophilic bacteria from different hot springs across the country. One of these sites is the Batu Kuwung hot spring in Banten. Neither DNA pol I nor thermophilic bacteria have been explored in this place to date. Therefore, the aim of this research is to clone the gene of DNA pol I from thermophilic bacteria in the Batu Kuwung hot spring.  This research hypothesizes that thermophilic bacteria can be isolated from the hot spring and be used to clone its DNA pol I gene to E. coli. This research is the initial step for DNA pol I production from indigenous thermophilic bacteria from the Batu Kuwung hot spring in future research. 


Conclusion

    From the results of this study, it can be concluded that the recombinant plasmid contains the desired gene of interest that is confirmed by digestion using the same restriction enzymes in making of the recombinant plasmid to identify the same restriction areas from gene cloning. The products of the digestion are also consistent with the theoretical gene size ( ), despite the slight shift of the bands’ positions. The result is further supported by the sequencing, where the approximately first and last  sequences of the gene of interest inside the recombinant plasmid are consistent with the theoretical nucleotide sequence and correlate with the amino acid sequence. Additionally, the SDS-PAGE result protein expression assay suggests a significant overexpression of proteins at around  where the target protein has a molecular weight of . It is highly encouraged to scale up the protein expression and use a nickel column to purify the His-tagged recombinant protein for further characterization in future research.

Acknowledgement

We acknowledge the financial support from The Directorate of Research and Community Service (DRPM) Universitas Indonesia through Grant of Indexed Publication (Publikasi Internasional Terindeks - PIT 9) No. NKB- 0043/UN2.R3.1/HKP.05.00/2019.

References

Akhmaloka, Pramono, H., Ambarsari, L., Susanti, E., Nurbaiti, S., Madayanti, F., 2008. Cloning, Homological Analysis, and Expression of DNA Pol I from Geobacillus thermoleovorans. International Journal of Integrative Biology, Volume 1(3), pp. 206–215

Aliotta, J.M., Pelletier, J.J., Ware, J.L., Moran, L.S., Benner, J.S., Kong, H., 1996. Thermostable Bst DNA Polymerase I Lacks a 3? ? 5? Proofreading Exonuclease Activity. Genetic Analysis - Biomolecular Engineering, Volume 12(5–6), pp. 185–195

Beyer, P., Al-Babili, S., Ye, X., Lucca, P., Schaub, P., Welsch, R., Potrykus, I., 2002. Golden Rice: Introducing the ?-Carotene Biosynthesis Pathway into Rice Endosperm by Genetic Engineering to Defeat Vitamin A Deficiency. The Journal of Nutrition, Volume 132(3), pp. 506S–510S

Cranenburgh, R.M., 2004. An Equation for Calculating the Volumetric Ratios Required in a Ligation Reaction. Applied Microbiol Biotechnol, Volume 65, pp. 200–202

Engelke, D.R., Krikos, A., Bruck, M.E., Ginsburg, D., 1990. Purification of Thermus Aquaticus DNA Polymerase Expressed in Escherichia coli. Analytical Biochemistry, Volume 191(2), pp. 396–400

Gomes, C.M., Faísca, P.F.N., 2019. Protein Folding: An Introduction. Springer. doi: 10.1007/978-3-319-00882-0_1

Hermansyah, H., Maresya, A., Putri, D. N., Sahlan, M. and Meyer, M. (2018) ‘Production of Dry Extract Lipase from Pseudomonas Aeruginosa by the Submerged Fermentation Method in Palm Oil Mill Effluent’, International Journal of Technology, 9(2), p. 325. doi: 10.14716/ijtech.v9i2.1511.

Li, P., Amenov, A., Kalendar, R., Abeldenov, S., Khassenov, B., 2017. Cloning and Purification of Large Fragment of DNA Polymerase I from Geobacillus stearothermophilus and it’s Application in Isothermal DNA Amplification. Biotechnology Theory and practice, Volume March 2017, pp. 1-12. doi: 10.11134/btp.1.2017.6

Maddocks, S., Jenkins, R., 2017. Understanding PCR?: A Practical Bench-top Guide. Academic Press

Sandalli, C., Singh, K., Modak, M.J., Ketkar, A., Canakci, S., Demir, I., Belduz, A.O., 2009. A New DNA Polymerase I from Geobacillus caldoxylosilyticus TK4: Cloning, Characterization, and Mutational Analysis of Two Aromatic Residues. Applied Microbiology and Biotechnology, Volume 84(1), pp. 105–117

Valones, M.A.A., Guimarães, R.L., Brandão, L.A.C., De Souza, P.R.E., De Albuquerque Tavares Carvalho, A., Crovela, S., 2009. Principles and Applications of Polymerase Chain Reaction in Medical Diagnostic Fields: A Review. Brazilian Journal of Microbiology, Volume 40(1), pp. 1–11

Zheng, W., Wang, Q., Bi, Q., 2016. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli. Protein Journal, Volume 35(2), pp. 145–153