Synthetic biology-based portable in vitro diagnostic platforms

Early diagnosis of infectious diseases represents powerful means to increase patient survival rate, avoid disease spreading, and decrease healthcare costs. Current Polymerase Chain Reaction (PCR)- and antibody-based diagnostic methods for detecting pathogens offer rapid analysis with highly accurate and specific results. However, those methods are still hampered by the need of sophisticated infrastructures and highly-skilled technicians, which limit the deployment in developing area. Synthetic biology with its rational and short design-to-production cycles has the potential to overcome those limitations. Here, we discuss two promising efforts for pathogen nucleic acids detection using synthetic biology approaches: Synthetic RNA-based and Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated (CRISPR/Cas)-based biosensors. The two systems were reported to show remarkable specificity and sensitivity on detecting and reporting the presence of pathogen via pathogen nucleic acid recognition with lower development and operational costs when compared to current PCR- and antibody-based diagnostic tools. Moreover, both systems can be applied to paper-based platforms which simplify the distribution and utilization in low resource-settings.Keywords: In vitro diagnostics, Synthetic biology, RNA-based biosensors, Toehold switch, CRISPR/Cas syste

Bio Prospecting Thermostable Enzymes-Producing Thermophiles From Indonesia

Thermostable enzymes-producing thermophilic microorganisms have been of industrial interest due to their ability to work optimally in high temperature which ensures high reaction efficiency and productivity,as well as reduces contamination risks. Efforts on isolating thermophilic microorganism with industrial potential have long been conducted in Indonesia, where there is large number of hot springs and geothermal area. Bacterial strains with the ability to produce thermostable industrial enzymes such as amylases, chitinase,lipase, protease, and xylanase have been isolated from various regions in Indonesia. This review is the first endeavor to collectively look into the researches on thermophilic microorganisms with industrial potential in Indonesia, their outcomes and proposed future directions based on the limitation of current studies

INDIGENOUS BACILLUS SPECIES ISOLATED FROM AEDES AEGYPTI LARVAE: ISOLATION, LARVICIDAL TOXICITY SCREENING, PHENOTYPIC CHARACTERIZATION, AND MOLECULAR IDENTIFICATION

Vector-borne diseases transmitted by mosquitoes are considered a significant public health problem worldwide. Aedes aegypti is one of the mosquito species responsible for transmitting these diseases. One environmentally friendly method of vector control is the use of microbial agents such as Bacillus species. This study aimed to explore investigate indigenous entomopathogenic bacteria of Bacillus species isolated from A. aegypti larvae. Larvae samples were collected from breeding sites of A. aegypti. All isolates underwent screening and affirmation confirmation tests to assess their larvicidal toxicity against A. aegypti larvae. Phenotypic characterizations and molecular identifications were conducted to determine the species of the Bacillus isolates based on similarity index and percent identity (%ID). Phylogenetic trees were used to compare the isolates with other Bacillus species. The results revealed 120 isolates of Bacillus species from A. aegypti larvae samples. Among them, three isolates (LS3.3, LS9.1, and LSD4.2) exhibited the highest larvicidal toxicity in the confirmation test, resulting in larval mortality rates of 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. Molecular identifications, showed that LSD4.2 had a 99.16% ID with Bacillus velezensis, LS3.3 had a 98.22% ID with Bacillus mojavensis, and LS9.1 had a 99.93% ID with Bacillus subtilis. These three bacteria from the Bacillus genus have been reported to offer significant benefits to humans

Peningkatan Ketahanan Terhadap salinitas Tinggi pada Tanaman Pangan Melalui Analisis dan Transplantasi Mikrobioma dari Tumbuhan Halofit Ekosistem Gumuk Pasir

SK Rektor Unair Nomor 672/UN3/202

Peningkatan Ketahanan Terhadap salinitas Tinggi pada Tanaman Pangan Melalui Analisis dan Transplantasi Mikrobioma dari Tumbuhan Halofit Ekosistem Gumuk Pasir

SK Rektor Unair Nomor:275/UN3/202

Enzymatic biotransformation of ginsenoside Rb1 by recombinant β-glucosidase of bacterial isolates from Indonesia

In this investigation, Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) together with its two transpeptidase-specialized mutants (N450D and N450Q) were employed for the biocatalytic synthesis of L-theanine. A high-pressure liquid chromatographic method coupled with ultraviolet detection (HPLC/UV) was used to quantify L-theanine content in the reaction mixture. Through a series of experiments, the perfect conditions for the biocatalytic synthesis were found to be an operational pH of 10.5, a reaction time of 4 h, L-glutamine and ethanolamine in a 1:2.4 M ratio, and enzymes at a working concentration of 25 μg/mL. In a batch process for N450D-mediated synthesis of L-theanine at 37 °C, a conversion rate of ~94% was achieved from 250 mM l-glutamine and 600 mM ethylamine. Successful synthesis of the desired product was further verified by mass spectrometry analysis. Conclusively, the experimental results suggest a high potential application in the production of L-theanine by BlGGT-mediated biocatalysis

Biosurfactant activity of indigenous Bacillus sp. ES4.3 isolated from endemic breeding sites of dengue hemorrhagic fever vector in Surabaya, East Java, Indonesia

Nafidiastri FA, Susetyo RD, Nurhariyati T, Supriyanto A, Geraldi A, Ni’matuzahroh, Fatimah, Salamun. 2021. Biosurfactant activity of indigenous Bacillus sp. ES4.3 isolated from endemic breeding sites of dengue hemorrhagic fever vector in Surabaya, East Java, Indonesia. Biodiversitas 22: 5375-5381. Bacillus spp. have shown the ability to results a variety of commercial bioactive compounds such as proteins, peptides, and lipopeptides (LPs). Some of the LPs produced by Bacillus spp. are surfactin, iturin, and fengicin. This study aimed to determine the name of the indigenous Bacillus sp. ES4.3, the biosynthesis surfactin gene, and the potential activity for biosurfactant produced by entomopathogenic Bacillus sp. ES4.3 isolated from endemic breeding sites of ??Dengue Hemorrhagic Fever Vector in Surabaya, East Java, Indonesia. Genomic DNA of Bacillus sp. ES4.3 was detected by isolating the DNA and visualizing it by electrophoresis. Furthermore, the 16S rRNA gene was amplified by the Polymerase Chain Reaction (PCR) method. The resulting nucleotide sequences were analyzed to find the relationship between Bacillus sp ES4.3 with another bacteria using MEGA version 6 software. Detection of biosynthesis surfactin gene was carried out by PCR method using srfAD primers. Analysis of the homology level of the surfactin gene was performed using the NCBI BLASTn and BLASTp genetic analysis program. The indigenous Bacillus sp. ES4.3 had 97.66% closeness to the species Bacillus velezensis FZB42 and the surfactin gene showed a 100% ID with the surfactin biosynthesis thioesterase SrfA-D gene on the Bacillus amyloliquefaciens group. The biosurfactant activity was indicated by the formation of clear zones, emulsions, and a decrease in surface tension in the values ??of 21.38 mN/m from the NB medium control and 33.74 mN/m from the distilled water control. The ability of B. velezensis ES4.3 to hemolyzed and reduce surface tension indicated the presence of biosurfactant that can disrupt stability and damage the midgut of Aedes aegypti. Thus, B. velezensis ES4.3 has the potential to be developed as a biocontrol in disease vectors

Enhanced Production of Fatty Acid Ethyl Ester with Engineered fabHDG Operon in Escherichia coli

Biodiesel, or fatty acid ethyl ester (FAEE), is an environmentally safe, next-generation biofuel. Conventionally, FAEE is produced by the conversion of oil/fats, obtained from plants, animals, and microorganisms, by transesterification. Recently, metabolic engineering of bacteria for ready-to-use biodiesel was developed. In Escherichia coli, it is produced by fatty acyl-carrier proteins and ethanol, with the help of thioesterase (TesB) and wax synthase (WS) enzymes. One of the foremost barriers in microbial FAEE production is the feedback inhibition of the fatty acid (FA) operon (fabHDG). Here, we studied the effect of biodiesel biosynthesis in E. coli with an engineered fabHDG operon. With a basic FAEE producing BD1 strain harboring tes and ws genes, biodiesel of 32 mg/L were produced. Optimal FAEE biosynthesis was achieved in the BD2 strain that carries an overexpressed operon (fabH, fabD, and fabG genes) and achieved up to 1291 mg/L of biodiesel, a 40-fold rise compared to the BD1 strain. The composition of FAEE obtained from the BD2 strain was 65% (C10:C2, decanoic acid ethyl ester) and 35% (C12:C2, dodecanoic acid ethyl ester). Our findings indicate that overexpression of the native FA operon, along with FAEE biosynthesis enzymes, improved biodiesel biosynthesis in E. coli

Potential biocontrol agent of indigenous Bacillus sp. EG6.4: Molecular identification, larvicidal toxicity, and mechanism of actions

Salamun, Susetyo RD, Nafidiastri FA, Zain RA, Sari RP, Geraldi A, Fatimah, Ni’matuzahroh. 2022. Potential biocontrol agent of indigenous Bacillus sp. EG6.4: Molecular identification, larvicidal toxicity, and mechanism of actions Biodiversitas 23: 5431-5438. This research was carried out for molecular identification, as well as the determination and mechanism of action of larvicidal toxicity of Bacillus sp. EG6.4 isolated from breeding sites of Aedes aegypti from Gresik, East Java, Indonesia. Bacillus sp. EG6.4 was a Gram-positive endospore-forming bacteria. Molecular species identification using 16S rRNA gene sequencing showed that isolate had 97.89% similarity with Bacillus mojavensis. The isolate showed larvicidal toxicity against A. aegypti larvae. The lethal concentration 50% (LC50) values ??at 24 and 48hours exposure were 8.99±1.01 ×107 cells/mL and 8.43±1.01 ×107 cells/mL, respectively, while lethal time 50% (LT50) value was 11.9±1.1 hours. Production of chitinolytic enzymes or biosurfactants, chitinolytic and hemolytic assays were conducted to determine the larvicidal mechanism. As a result, Bacillus sp. EG6.4 showed hemolytic, but not chitinolytic activity, indicating its potency to produce biosurfactants. Transmission Electron Microscopy (TEM) result showed that isolate had oval-shaped endospores located subterminal with massive-shape parasporal inclusions. The detection of srfA-D gene showed that isolate produced surfactin biosynthesis thioesterase. Thus, Bacillus sp. EG6.4 produced biosurfactant that potentially to be developed as a biocontrol agent for disease vectors and plant pathogens

Biosurfactant production of entomopathogenic Bacillus subtilis BK7.1, as potential biocontrol bacteria, isolated from Baluran National Park, East Java, Indonesia

Salamun, Susetyo RD, Ni’matuzahroh, Fatimah, Geraldi A, Supriyanto A, Nurhariyati T, Nafidiastri FA, Nisa’ N, Endarto. 2023. Biosurfactant production of entomopathogenic Bacillus subtilis BK7.1, as potential biocontrol bacteria, isolated from Baluran National Park, East Java, Indonesia. Biodiversitas 24: 1785-1792. Biosurfactants as biocontrol agents have received much attention for pest control and disease vectors. The research aimed to identify the species and genetic relationship, hemolytic activity, detect coding genes, and trial production of biosurfactants on various substrates of entomopathogenic Bacillus sp. BK7.1 isolated from natural soil in Baluran National Park, East Java, Indonesia. Biosurfactant screening was carried out by testing hemolytic activity, surface tension, and emulsification activities, detecting coding genes of biosurfactant biosynthesis, and testing biosurfactant production in various substrates. The results of the molecular identification by amplifying the 16S rRNA gene using the Polymerase Chain Reaction (PCR) method for Bacillus sp. BK7.1 has a genetic similarity of 98.68% with B. subtilis subsp. inaquosorum strain BGSC 3A28. Screening showed positive hemolytic activity results, reduced surface tension, increased emulsification activities, and the production of biosurfactant in glucose, glycerol, and molasses substrates. The PCR results showed that Bacillus sp. BK7.1 had srfAA and srfAD genes encoding surfactin biosynthesis, giving it the potential to produce bioinsecticide compounds. Based on these studies, the indigenous entomopathogenic B. subtilis BK7.1 can be developed as environmentally friendly microbial bioinsecticides for pest control and disease vectors