Published at : 20 Nov 2020
Volume : IJtech
Vol 11, No 5 (2020)
DOI : https://doi.org/10.14716/ijtech.v11i5.4311
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 |
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
DNA pol I; DNA polymerase; Escherichia coli; Geobacillus thermoleovorans SGAir0734; Thermophilic bacteria
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
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.
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 (
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.
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