Indonesian Journal of Biotechnology
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Cloning and Expression of hGAD65 Gene in E. Coli BL21
No full textThe aim of this study is to construct the hGAD65 gene and to identify the hGAD65 clone by using PCR & RFLP. The samples were derived from normal person & DM patient’s blood. Blood DNA was isolated by salting out method and then amplified by PCR with a pair of specific primer, GAD65-F-BamH1-807 & GAD65-R-Xho1-945. The PCR-product was cloned into vector pET-28a and the pET28a-hGAD65-clone was transformed into E.coli BL21 competent cells. The pET28a-hGAD65-clone was confirmed by PCR and RFLP by BamH1 & XhoI. The PCR product of pET28a-hGAD65-clone was one band of 159bp and has two bands 5.3 kb and 159 bp by RFLPwith both restriction enzymes. The GAD65 protein is expressed in 65kD of pET28a-hGAD65-clone. PET28a-hGAD65-clone was able to recognize by gold standard monoclonal antibody specifically. These results indicated that the hGAD65 gene inserted into pET28a properly and provided the GAD65 protein expression. Key words: hGAD65, PCR, pET-28a, RFL
The Application of Polymerase Chain Reaction – Restriction Fragment Polymorphisms (PCR-RFLP) to Determine Genetic Diversity of Madura Cattle in Sapudi Island
No full textThe aim of this study was to determine genetic diversity of Madura cattle using Polymerase Chain Reaction – Restriction Fragment Length Polymorphisms (PCR-RFLP) analysis of the cytochrome b (cytb) gene. Samples used for the experiments were blood of 43 cattle that consist of 15 cattle obtained from Madura Island, 23 cattle from Sapudi Island, and 5 Limousin-Madura (Limura) cattle. A fragment of 464 base pair of cytb gene was amplifi ed by forward primer L14735 and reverse primer H15149. The PCR product was digested with TaqIand HinfI restriction enzymes to identify genetic patterns. Data of PCR-RFLP showed two haplotypes, that were A and B, in cattle obtained from both Madura Island and Sapudi Island. The frequencies of haplotype A and B of cattle from Sapudi Island were 69.57% and 30.47%, respectively. More diverse frequencies were observed in cattle obtained from Madura Island, where haplotype A and B were 86.67% and 13.33%, respectively. In this experiment, Limura cattle had only haplotype A. As a conclusion, PCR-RFLP of the cytb gene had been able to determine a genetic diversity of Madura cattle. Key words: Genetic diversity, Madura cattle, haplotype
Characterization of Streptomyces spp. Producing Indole-3-acetic acid as Biostimulant Agent
No full textTwenty six isolates of Streptomyces spp. obtained from Cyperus rotundus L. rhizosphere were tested forability to produce indole-3-acetic acid (IAA) in yeast malt extract (YM) medium containing 2 mg/mL tryptophan.Screening of the isolates for ability to produce IAA was carried out by adding Salkowski reagent in bacteriaculture and was measured quantitatively by spectrophotometer at λ 530 nm. Thin Layer Chromatography (TLC)method was used to determine IAA. To ensure the IAA production in Streptomyces isolates, gene involved inIAA biosynthesis was detected by amplifying Tryptophan Monooxigenase (iaaM) gene. The study of the effectof tryptophan on the production of IAA was measured at different concentrations of tryptophan (0, 1, 2, 3,4, 5 mg/mL) in the bacterial culture. The result showed that there were two Streptomyces spp. isolates whichcould produce IAA, namely the isolates of Streptomyces sp. MS1 (125.48 μg/mL) and Streptomyces sp. BR27(104.13 μg/mL). The TLC result showed that the compound in both isolates was identifi ed to be IAA. Theamplifi cation results showed that iaaM gene was detected in both isolates. This results indicated that the IAMpathway is predicted involved in the biosynthesis of IAA in the selected isolates. Both of the isolates were ableto produce IAA after 24 h incubation and the highest production was at 120 h incubation with the concentrationof tryptophan was 2 mg/mL dan 1 mg/mL, respectively. Therefore, it is concluded that Streptomyces spp.isolates are able to produce IAA and potentially to be utilized as biostimulat agent. Keywords: Streptomyces spp., indole-3-acetic acid (IAA), indole-3-acetamide (IAM), Tryptophan Monooxigenasegene (iaaM
The Development of Pathogenicity of Avian Influenza Virus Isolated from Indonesia
No full textHighly pathogenic avian infl uenza outbreak in Indonesia has been reported in various poultry due toH5N1 subtype. The presence of multiple basic amino acids within the cleavage site of HA glycoprotein hasbeen identifi ed to be associated with the pathogenicity of avian infl uenza virus. The study was retrospectivestudy which was designed to characterize the cleavage site and fusion site region of haemagglutinin gene ofAIV isolated from various poultry in 2003 to 2013. Isolation, Identifi cation and propagation were carried outto collect viral stock. For virus detection, reverse transcriptase PCR (RT-PCR) method on H5 and N1 genefragment was performed. All of RT-PCR HA gene positive products were sequenced for further nucleotideanalysis and to determine the nucleotide composition at the targeted fragment. The results are all AIV isolateswere identifi ed as H5N1 subtype. The sequence analyses revealed some motives of basic amino acid motivethat were classifi ed as highly pathogenic avian infl uenza virus. Further analyses on fusion domain of all AIVisolated during the period 2003 to 2013 showed conserved amino acid. Keywords: avian infl uenza, haemagglutinin, cleavage site, basic amino acid, fusion sit
Purification and Characterization of Protease From Bacillus sp. TBRSN- 1
No full textPotato Cyst Nematode (PCN), Globodera rostochiensis, is one of the important potato’s pests and causedeconomic looses up to 70% in the several centrals of potato plantations in Indonesia. PCN’s shell componentof egg shell containing chitin (inner layer) and viteline/ protein (outer layer). The purpose of this researchwas to purify of protease Bacillus sp. TBRSN-1, isolate from tomato’s rhizosfer in Yogyakarta province. Thepurifi ed protease could be used for cutting the life cycle of PCN. Results showed that Bacillus sp. TBRSN-1could produce extracellular protease and purifi cation using DEAE-cellulose ion-exchange chromatographyand Sephacryl S-300 gel fi ltration chromatography resulted in specifi c activity 4.31 fold and 1.68% recovery.Analysing using SDS-PAGE 12.5% and molecular weight 48.1 kDa. Km and Vmax values of the protease forcasein substrate were 7.83 mg/ml and 4.03 μg/h, respectively. The optimum activity at the temperature30oC and pH 7.0. Keywords : protease, purifi cation, indigenous Bacillus sp. TBRSN-
Genotyping of Rotavirus by Using RT-PCR Methods
No full textThere is a great diversity of rotavirus genotypes circulating worldwide, with dominant genotypes changing from year to year. Rotavirus genotyping was performed by using reverse transcription PCR with type-specifi c-primers. Since rotavirus is a RNA virus that has high mutation rate, there was a possibility of technical diffi culty in genotyping due to mutation in the primer binding sites. During Indonesian rotavirus surveillance study 2006-2009, it was reported that 17% of samples subjected for G type and 21% of samplessubjected for P type were untypeable. The objective of this study was to identify genotypes of the samples that were untypeable previously using RT-PCR based on the method described by Das et al. (1994) and Gentsch et al. (1992). There were 30 samples subjected to G type and 61 samples subjected to P type to be re-typed using method described by Gouvea et al. (1990) and Simmond et al. (2008) for G and P typing, respectively. By using another set of primer, the genotype of all samples was identifi ed. This study highlights the importance of a constant reconsideration of primer sequences employed for the molecular typing of rotaviruses. Key words: rotavirus, G typing, P typin
Apolipoprotein E as Risk Factor for Coronary Heart Disease
No full textAllelic variation of apolipoprotein E (apo E) has been shown to infl uence the concentrations of total cholesterol and low density lipoprotein cholesterol (LDL-C) and considered to play a role as one of risk factors for coronary heart disease (CHD). The aim of this study was to examine the relationship between Apo E polymorphism and the risk of CHD. Blood samples were collected from 33 CHD patients in Dr. Sardjito Hospital Yogyakarta, and 38 apparently healthy control individuals in a cross sectional study. The common allelic variants of ApoE were screened employing polymerase chain reaction and restriction fragment length polymorphism. The results obtained were analyzed by t-test and signifi cantly different if p <0.05 and risk factor was calculated by odd ratio. Frequency of ApoE ε2, ε2 and ε4 alleles in CHD patients were 12.1%, 69.7% and 18.2% while in controls were 18.4%, 72.4% and 9.2% respectively. Dyslipidemia condition was a strongrisk factor for CHD. By controlling lipid profi le and applying multifactorial statistic analysis, it was shown that ε4 gene carrier was the risk factor for CHD, but not in triglyceride level, whereas ε2 carrier gene was not the risk factor for CHD. Dislipidemia was the risk factor for CHD and ApoE ε4 gene carrier was the risk factor for CHD. Key words: apolipoprotein E, ApoE ε4 gene carrier, coronary heart disease, dyslipidemia
A Novel Variant of HOXA10 gene, Ser19Cys, among Patients with Endometriosis and its Relationship with the Severity of the Disease
No full textEndometriosis is a gynecological disease associated with inherited genetic traits. HOXA10 gene whichis expressed in uterine plays an important role in the pathogenesis of endometriosis. The protein affects thedevelopment of pinopodes as a biomarker of endometrial receptivity in endometriosis.The aim of this study isto examine if there is a mutation or polymorphism within HOXA10 gene among patients with endometriosis.Thirty twopatients and 32 healthy women were recruited as subjects of this study. The exon 2 of HOXA10which covers most of coding region was amplifi ed using PCR. The presence of a mutation or polymorphismwas detected by direct seguencing. The distribution of genotype and allele was analyzed using Chi square test with p<0.05 is considered as signifi cantly different. A novel heterozygous variant within exon 2 of HOXA10 which substitute an adenine into thymine was detected at base position 55. This missense alteration changed amino acid serine to cystein (Ser19Cys). Interestingly, this variant was detected in 12 endometriosis cases (38%) but none in control. Patients carry HOXA10 Ser19Cys variant were associated with dismenorea and more frequent in stage I endometriosis. The role of this variant in the function of HOXA10 protein and frequency among Indonesians need to be clarifi ed. We found a novel heterozygous HOXA10 gene variant, Ser19Cys.The genotype frequency is 38% among endometriosis patients but none in control. This variant found in patient with dismenore and endometriosis stage 1. Key words: HOXA10 gene, endometriosis, Ser19Cys polymophis
Stachybotrys chartarum: A Novel Biological Agent for The Extracellular Synthesis of Silver Nanoparticles and Their Antimicrobial Activity
No full textMicrobial assisted biosynthesis of nanoparticles is a rapidly progressing area of nanobiotechnology. Inthis paper Stachybotrys chartarum assisted extracellular synthesis of silver nanoparticles (AgNPs) is reportedwhen challenged with 1mM silver nitrate (AgNO3). The characterization of AgNPs was carried out visualobservation and UV-Vis spectrophotometry. Further analysis carried out by Fourier Transform InfraredSpectroscopy (FTIR), provides evidence for the presence of proteins as capping agent, which helps in increasingthe stability of the synthesized AgNPs. Transmission Electron Microscopy (TEM) investigations confi rmedthat AgNPs were formed. The synthesized silver nanoparticles were found in the range of 65-108 nm. Finally,the antimicrobial susceptibility of AgNPs synthesized was investigated which exhibited more potent activityagainst bacteria than fungi compared with using silver nitrate at concentration 1mM. Keywords: Antimicrobial activity, Stachybotrys chartarum, Silver nanoparticle
A Study on Production of Poly-β-Hydroxybutyrate Bioplastic from Sago Starch by Indigenous Amylolytic Bacteria
No full textBacillus sp. PSA10 and Bacillus sp. PPK5 were two indigenous strain amylolytic bacteria from SoutheastSulawesi that have ability to produce bioplastic poly-β-hydroxybutyrate (PHB) from sago starch. The study wasattempted to determine the mechanism of PHB production by bacteria amylolytic was grown on sago starchcontainingmedia. Two amylolytic bacteria i.e. Bacillus sp. PSA10 and Bacillus sp. PPK5 was grown for 168 hin a mineral salts medium with sago starch as carbon source. Growth of amylolytic bacteria was monitoredby cell dry weight. Extraction of PHB was done by N-hexane acetone-diethyl ether method and PHB contentwas quantifi ed with UV spectrophotometer at 235 nm. Glucose level was determined by using kit of glucoseGOD 10” and was quantifi ed with spectrophotometer at 500 nm. Sago starch concentration was determinedby phenol method using specthrophotometer at 490 nm. The result of the study showed that Bacillus sp.PSA10 was produced PHB up to 66,81 % (g PHB/g cell dry weight) at 48 h and Bacillus sp. PPK5 up to 24,83% (g PHB/g cell dry weight) at 84 h. Bacillus sp. PSA10 has ability to converse sago starch to be PHB directlywithout glucose accumulation in the media, whereas Bacillus sp. PPK5 have to accumulate glucose as productof sago starch hydrolysis to produce of PHB. PHB synthesis by Bacillus sp. PHB production on sago starchof the Bacillus sp. PSA10 was found to be growth-associated whereas Bacillus sp. PPK5 was found to be nongrowth-associated. Therefore, two indigenous amylolytic bacteria were having of difference in biosynthesismechanism of PHB in sago starch medium and their characteristics of PHB synthesis should be consideredin developing cultivation methods for the effi cient production of PHB. Keywords : Production, PHB, Amylolytic bacteria, Sago starch