Indonesian Journal of Biotechnology
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The effectivity of thidiazuron and 1‐naphthaleneacetic acid on somatic embryo induction in transgenic Dendrobium phalaenopsis Fitzg. carrying 35S::GR::AtRKD4
No full textDendrobium phalaenopsis Fitzg. (also known as the Larat orchid) is an endemic orchid from Larat Island, Eastern Indonesia. Its beautiful flowers mean that many plants are taken for commercial purposes, leading to the rapid decline of populations in their natural habitats. The objectives of this study were to determine which organs of the transgenic Larat orchid carrying the 35S::GR::AtRKD4 construct, together with which concentrations of the plant growth regulators (PGRs) auxin and cytokinin, are suitable for the induction of somatic embryos (SEs). In this study, the AtRKD4 gene in Larat orchids was confirmed using PCR with specific primers for the AtRKD4 and HPT genes. Thidiazuron (TDZ) (1, 3 and 5 mg/L) in combination with 1‐naphthaleneacetic acid (NAA) (0.5 and 1 mg/L) were used on new phalaenopsis (NP) medium to induce SEs from leaves, pseudobulbs and roots. The AtRKD4 transgenes were detected as being stably integrated into the DNA genome of transformant plants using specific primers for AtRKD4 and HPT genes, and positive results were obtained using actin gene primers as internal controls for PCR. Pseudobulbs produced 19 to 20 SEs from 108 pseudobulb explants (89–100%), a higher number than produced in explants of the other organs studied. Among the PGR treatments, the best results were obtained in NP medium supplemented with a combination of 1 mg/L TDZ and 1 mg/L NAA, 100% of the explants of which produced SEs (2.11 ± 1.36). No significant difference was found between the morphology of the SEs produced from the non‐transformant Larat orchid pseudobulb explants and the 35S::AtRKD4 carrier transformant
Tetra‐primer amplification refractory mutation system (ARMS) PCR used to detect 3’UTR rs1948 mutation in CHRNB4
No full textRs1948 A>G is a single nucleotide variation (SNV) in the 3’‐UTR of CHRNB4. Genotyping the synonymous CHRNB4 rs1948 may be useful in identifying a lung cancer susceptibility gene. The study aimed to develop a simple and easy tetra‐primer amplification refractory mutation system (ARMS PCR) for CHRNB4 rs1948. The following steps were taken to optimize tetra‐primer ARMS PCR: 1) determining the gene sequence and position of a single mutation; 2) developing outer and inner primers; 3) amplification of target gene fragments via PCR using an outer primer; 4) genotyping PCR product using Sanger sequencing; 5) determining the optimal annealing temperature and PCR cycle; 6) determining optimal outer and inner primer ratio; and 7) testing the reproducibility of the PCR program and final validation with Sanger sequencing. Genotype (PCR result) was visualized with 3% agarose gel electrophoresis. Optimum condition was determined as annealing temperature of 64.8 ºC and 35 cycles, outer and inner primer ratio of 1:6, and DNA volume of 3 µL. Sanger sequencing confirmed the results of the tetra‐primer ARMS PCR and it was shown that ARMS PCR was able to identify three different variants of CHRNB4 rs1948
Pantoea agglomerans, Klebsiella pneumoniae, and Shigella flexneri isolated from the Cisadane River as multiresistant bacteria to copper and dyes
No full textCopper pollution in Cisadane is a serious environmental issue that needs to be resolved immediately due to its negative impacts on river ecosystems. Bioremediation utilising indigenous bacteria offers excellent potential to restore copper‐contaminated river water. This study aimed to obtain indigenous copper‐resistant bacteria isolated from the Cisadane River as copper bioremediation agents. Bacteria from Cisadane River water samples were isolated by the spread plate method on Luria Bertani medium containing 3 mM CuSO4. Resistance was determined based on the minimum inhibitory concentration value, while copper concentration was measured using an atomic absorption spec‐ trophotometer. The results presented a total of 13 bacterial isolates with a minimum inhibitory concentration of up to 8 mM CuSO4. Sequence alignment analysis was performed on three selected copper‐resistant bacteria, i.e. isolate IrCis1, IrCis4 and IrCis13, which were identified as Pantoea agglomerans, Klebsiella pneumoniae and Shigella flexneri based on 16S rRNA, respectively. Each isolate accumulated copper at 1.19 mg, 1.34 mg and 0.92 mg/g DW of cells, with copper biosorption potentials of 73.74%, 70.17% and 67.73%, respectively. In conclusion, P. agglomerans strain IrCis1, K. pneu‐ moniae strain IrCis4 and S. flexneri strain IrCis5 isolated from the Cisadane River can be used as copper bioremediation agents
Characterization of the urogenital microbiome in patients with urinary tract infections
No full textStandard microbiological culture techniques can only identify a fraction of the urogenital microbiome. Meanwhile, identifying and characterizing infectious microorganisms are very important for the success of diagnosis and treatments, especially for Urinary Tract Infection (UTI) patients. This study aimed to characterize the urogenital microbiome of UTI patients using 16S rRNA gene sequencing. We sequenced two pooled DNA samples from voided urine of UTI patients (21 females and 13 males). To determine the structure and composition of taxa in the samples, 16S rRNA gene sequencing was performed using the Illumina Mi‐Seq paired‐end platform. The most abundant genera were Burkholderia‐Caballeronia‐Paraburkholderia (71%) followed by Prevotella (33%), Escherichia‐Shigella (24%), Klebsiella (23%) and Sneathia (10%). The female microbiome was dominated by Prevotella bivia (28%), Escherichia coli (24%), Sneathia sanguinegens (7%) and Klebsiella pneumoniae (4%). On the other hand, the male microbiome was dominated by K. pneumoniae (23%) and E. coli (2%). K. pneumoniae and E. coli were the most abundant species found in both microbiomes. The 16S rRNA gene sequencing used in this study successfully uncovered the composition of the urogenital microbiome, which might not have been possible with conventional culture methods
Increased activity of sugarcane sucrose‐phosphate synthase in transgenic tomato in response to N‐terminal truncation
No full textSucrose‐phosphate synthase (SPS) is a key enzyme catalyzing the formation of sucrose‐6‐phosphate through the transfer of uridine‐diphosphate glucose (UDP‐G) as a donor to fructose‐6‐phosphate (F6P) as an acceptor. Plant SPS consists of three main domains: N‐terminal, glycosyltransferase, and C‐terminal domains. Among these, the N‐terminal domain is involved in regulating the allosteric activator glucose‐6‐phosphate (G6P). This study was directed toward determining the regulation and characterization of N‐terminal truncated SPS in transgenic tomato. In this study, the N‐terminal truncated mutant of sugarcane SPS (ΔN‐SoSPS1) and full‐length sugarcane SPS (FL‐SoSPS1) were expressed into tomato plants to verify the functional role and importance of the N‐terminal domain in plant SPS. Overexpression of ΔN‐SoSPS1 led to an up to 3‐fold increase in the specific activity of SPS compared to non‐transformant plants (WT), while the specific activity of ΔN‐SoSPS1 was higher than FL‐SoSPS1 in transgenic tomato plants. Unlike WT and FL‐SoSPS1, the ΔN‐SoSPS1 mutant was not allosterically regulated by G6P. These results indicated that deletion of the N‐terminal domain promotes the loss of allosteric activation by G6P and increases binding affinity between enzyme and substrate
Purification and characterization of thermostable alpha‐amylase from Geobacillus sp. DS3 from Sikidang Crater, Central Java, Indonesia
No full textAmylases are considered the most essential enzymes in biotechnology since they are widely utilized in the textile, food processing, and detergent industries. It is necessary to explore extracellular enzymatic activity in several microorganisms to discover a new potential application from amylases. In a previous study, thermophilic bacteria Geobacillus sp. DS3 isolated from Sikidang Crater, Dieng Plateau, Central Java, Indonesia showed amylase activity in starch medium at 70 °C. This study aimed to purify and characterize the thermostable alpha‐amylase from Geobacillus sp. DS3. The alpha‐amylase was produced and purified using ammonium sulfate and DEAE Sephadex A‐25 column. The enzyme activity was determined using the 3,5‐dinitrosalicylic acid (DNS) method. Geobacillus sp. DS3 optimally produced the alpha‐amylase at 60 °C for 15 h. The alpha‐amylase exhibited high enzymatic activity in 40–60% saturated ammonium sulfate extract. The molecular weight of the enzyme was estimated to be 58 kDa. The thermostable alpha‐amylase showed activity at the optimum temperature of 50 °C in 200 mM sodium phosphate buffer pH 7.0. The enzyme was inhibited by EDTA, PMSF, 2‐ME, and mostly by HgCl2. The Km and Vmax of the pure enzyme were 235.43 mM and 1428.57 U/mL, respectively. The result suggested that the purified thermostable alpha‐amylase from Geobacillus sp. DS3 offers potential application in areas of the food industry, such as the bakery industry
Increased serial levels of platelet‐derived growth factor using hypoxic mesenchymal stem cell‐conditioned medium to promote closure acceler‐ ation in a full‐thickness wound
No full textThe healing process of a full‐thickness wound involves a complex cascade of cellular responses to reverse skin integrity formation. These processes require growth factors, particularly platelet‐derived growth factor (PDGF). Conversely, hypoxic mesenchymal stem‐cell‐conditioned medium (HMSC‐CM)‐contained growth factors notably contribute to acceleration of wound healing. This study aims to investigate the role of HMSC‐CM in controlling the serial levels of PDGF associated with accelerated wound closure in full‐thickness wounds. Twenty male Wistar rats with full‐thickness wounds were developed as animal models. The animals were randomly assigned to four groups, comprising two treatment groups (treated using HMSC‐CM at a high dose as P1 and at a low dose as P2), a control group (administration of base gel), and sham group (healthy group). PDGF levels were examined using an enzyme‐linked immunosorbent assay. Using ImageJ software, wound closure percentages were determined photographically. The study showed that there was a significant increase in PDGF levels on days 3 and 6 after HMSC‐CM treatment, followed by a decrease in PDGF levels on day 9. In line with these findings, wound closure percentage also increased significantly on days 6 and 9. In the rat model, HMSC‐CM administration may promote acceleration of wound closure by increasing serial PDGF levels in the full‐thickness wound
Effect of galangal essential oils on rumen microbial population and biodiversity on in vitro rumen fermentation
No full textThe study aimed to evaluate the effect of administering galangal essential oil (EO) on the abundance of rumen bacteria using the 16s rRNA method. The treatments included a control (no EO addition), galangal EO (30, 60, 120 µL), and cineole (5 µL). The treatments were assessed using a 48‐hour in vitro batch culture of rumen fluid containing a 60:40 ratio of forage to concentrate. For amplification of the prokaryotes (bacteria and archaea) in region V4, 16s rRNA primer 5’GTGCCAGCMGCCGCGTAA, GGACTACHVGGGTWTCTAAT3’ was employed. The data for rumen microbial abundance were analysed descriptively, while the data for rumen microbial diversity were obtained from the report on the Next Generation Sequencing Method. The microbial composition of each sample was tested for operational taxonomic units (OTUs) with a 97% identity rate on a valid label. The 16S rRNA gene sequencing yielded a total of 3,977 OTUs. Adding galangal and cineole EOs resulted in the same variation of the Shannon index. The population index (chao1 index) was highest when 60 µL of galangal EO was added, compared to 30 and 120 µL of galangal EO and cineole. In addition, providing 60 µL of galangal EO decreased the abundance of Prevotella ruminicola compared to the control and cineole doses. The addition of galangal EO also led to a decline in the number of Methanobacteriales. The population of the fibre‐degrading bacteria group (Ruminococcus albus and Ruminococcus flavefaciens) was higher in a dose of galangal EO than the control and cineole. Therefore, it can be concluded that the effective dose of galangal EO, i.e. 60 µL/300 mg (DM feed) in vitro, can reduce the abundance of Prevotella bacteria and methanogens
Isolation and characterization of α ‐amylase encoding gene in Bacillus amyloliquefaciens PAS
No full textAmylolytic bacteria are a source of amylase, which is an essential enzyme to support microalgae growth in the bioreactor for microalgae culture. In a previous study, the highest bacterial isolate to hydrolyze amylum (namely PAS) was successfully isolated from Ranu Pani, Indonesia, and it was identified as Bacillus amyloliquefaciens. That bacterial isolate (B. amyloliquefaciens PAS) also has been proven to accelerate Chlorella vulgaris growth in the mini bioreactor. This study aims to detect, isolate, and characterize the PAS’s α‐amylase encoding gene. This study was conducted with DNA extraction, amplification of α‐amylase gene with polymerase chain reaction (PCR) method with the specific primers, DNA sequencing, phylogenetic tree construction, and protein modeling. The result showed that α‐amylase was successfully detected in PAS bacterial isolate. The α‐amylase DNA fragment was obtained 1,468 bp and that translated sequence has an identity of about 98.3% compared to the B. amylolyquefaciens α‐amylase 3BH4 in the Protein Data Bank (PDB). The predicted 3D protein model of the PAS’s α‐amylase encoding gene has amino acid variations that predicted affect the protein’s structure in the small region. This research will be useful for further research to produce recombinant α‐amylase
Unfolded protein response in rice (Oryza sativa L.) varieties with different level of salt stress tolerance
No full textPlants activate the unfolded protein response as part of cellular adaptation, thereby maintaining the endoplasmic reticulum homeostasis during external stresses exposure. In this study, we examined the relationship between the degree of salt tolerance and unfolded protein response-related gene expression in India salt-tolerant Pokkali and INPARI 35 varieties compared to the Indica salt-sensitive counterpart IR64 and INPARI 4 varieties. Our result showed that the salt tolerance of Pokkali and INPARI 35 had been confirmed by their higher survival rate, higher chlorophyll content, lower electrolyte leakage, and lower H2O2 and malondialdehyde content under salt stress conditions. Furthermore, the expression of unfolded protein response genes was highest in INPARI 35, whereas IR64 and INPARI 4 exhibited low gene induction during endoplasmic reticulum stress conditions. Among the four examined varieties the salt tolerant Pokkali surprisingly showed the lowest induction of all examined unfolded protein response-related genes. These results indicated the possibility that unfolded protein response supports the rice plant for adapting to the saline environment