Hussin, Nurul Akmar
(2013)
Identification, Cloning And Expressing
Of Dna Polymerase Producing
Thermophile From Geothermal Water
In Malaysia.
Masters thesis, Universiti Sains Malaysia.
Abstract
Aerotolerant anaerobe, Gram-positive with long rod in shape thermophilic bacteria
was successfully isolated from Ulu Legong Hot Spring, Kedah, Malaysia in this
study. The thermophilic bacteria was successfully isolated from the sediment sample
in this study and denoted as 3UL which showed the best growth and well adapted to
the laboratory condition compared with other isolates at temperature ranged between
45 to 80oC. Hence, this microbe was chosen for further study. A set of universal
primers (F_UNI16S and R_UNI16S) were used to amplify the 16S rRNA gene
sequences for ribotyping identification methods. Its 16S rRNA gene sequences (1454
nucleotides) showed very high homology (100%) with Anoxybacillus sp. DR04. The
16S rRNA gene sequence for 3UL has been deposited into “Genbank Data Library”
and assigned the accession number JQ951796. Genomic DNA from the isolate was
extracted and was used to amplify DNA polymerase I gene sequences. The NREAF2
and XREAR_Fxa were forward and reverse primers used for the DNA polymerase
amplification with restriction sites NcoI for forward and XhoI for reverse. The gene
was 2,628 bp long and encodes a protein of 876 amino acids in length. The enzyme
has molecular mass of 99 kDa and showed sequence homology with DNA
polymerase I (94%) from Anoxybacillus sp., (75%) Geobacillus sp., and (74%)
Bacillus sp. The gene was over expressed in Escherichia coli BL21 (DE3) by using
pET28a(+) as expression vector with his-tag at the C-terminus.
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