Sanger Sequencing Customer Publications

The utilisation of enzymes in the industry has brought numerous benefits and advantages to production processes. Enzymes serve as biocatalysts, efficiently catalyzing reactions and hydrolysis in biochemical processes. However, there are challenges in applying enzymes in the industry, particularly concerning enzyme stability. The obstacle encountered in the production processes involv-ing industrial enzyme applications is the low stability of enzymes when used at high temperatures. Heat-sensitive enzymes undergo damage or denaturation. Thermophilic microorganisms are chosen because they hold the potential to produce thermophilic enzymes. The thermophilic enzymes exhibit better heat stability compared to other enzymes, making them an effective alternative for future industrial production processes. This study aims to isolate thermotoler-ant bacteria from Nglimut Hot Spring sediment, screen for cellulase and amylase-producing isolates, and molecularly identify the best isolate using 16S rRNAbarcode. The results show that 22 bacterial isolates were found in the sediment of a hot spring; TS-14 was the best isolate in producing amylase, with the highest average amylolytic index of 2.38, whereas TS-15 had the highest cellulolytic index of 2.11. Based on 16S rRNA identification, TS-14 showed an homological identity of79% with Bacillus amyloliquefaciens, while TS-15 had a 100% homological identity with Bacillus licheniformis.These re-sults were important as the first step of screening bacterial potential to pro-duce thermophilic enzymes that could be applied in the downstream pro-cessing in future industrial and biotechnology companies.

The presence of ferritin protein in rice plants indicates a regulatory response to environmental iron stress. It prevents the destructive effects of the chain reaction resulting from Fe2+accumulation in the cell. This study aimed to detect and identify the location of the ferritin gene (OsFER) in eight local rice varieties in Indonesia. OsFER2, as the target gene was amplified using PCR, visualized by electrophoresis, and then sequenced. The sequencing results wereanalyzed using DNA Baser, BioEdit, and ClustalX2. The predicted proteins were visualized using the SWISS–MODEL server, Rice Genome Annotation Project Database, and chromosome map tools. The results show that all rice varieties studied have 100% alignment similarity and OsFERcharacteristics on chromosome 11 at LOC_Os11g01530 and chromosome 12 at LOC_Os12g01530, which have striking differences. The complete protein structure of the complex is found on chromosome 12, while only a portion of alpha–helix ferritin is found on chromosome 11. Differences in the position of the ferritin gene at different chromosomes impact the functional changes in the ferritin protein as an iron homeostasis key role. The two genes show different characteristics and are located at different genomic positions, suggesting a potential regulatory impact on the level of iron stress resistance in Indonesian rice varieties.

Advances in fermentation technology, genetic engineering, and enzyme application technology have increased the use of enzymes. Enzymes can be produced by utilizing a source of microorganisms such as bacteria. Proteolytic bacteria or protease enzyme-producing bacteria are found in foods or plants that contain protein such as the brown seaweed Hydroclathrus sp. This study aimed to obtain protease-producing bacterium associated with marine algae Hydroclathrus sp. from waters around Hoga Island of Wakatobi District and identify the organism based on its 16S rRNA gene sequence. Isolation of bacteria from algae samples was carried out with nNutrient aAgar (NA) media, while proteolytic bacteria selection was carried out on sSkim mMilk aAgar (SMA) media. The bacterial isolates producing proteolytic-clear zone on SMA media were then identified targeting the 16S rRNA gene using the PCR (Polymerase Chain Reaction) method with 27F-1492R primers. Based on the isolation results, there were 3 unique colonies of bacteria that could be cultured from algae samples and coded HIHA-1 to HIHA-3 (HIHA stands for Hoga Island Hydrolathrus macroalgae). The selection process for protease-producing bacteria on SMA media resulted in 1 isolate of proteolytic bacteria, namely HIHA-1. Molecular identification by PCR on HIHA-1 isolate resulted in a single DNA band on the electrophoresis gel sized ~1500 bp. The sequencing results showed a DNA sequence with the size of 1421 bp sharing the highest similarity with the bacterium Exiguobacterium aestuarii strain TF-16 (homology level of 99,93%). In conclusion, the proteolytic bacterial isolate HIHA-1 associated with marine brown algae Hydroclathrus sp. was obtained and identified as Exiguobacterium aestuarii strain HIHA-1.

Gaharu bouya oil obtained from distillation of the woods from Gonystylus genus has attracted essential oil industry interest. However, the information about gaharu bouya essential oil profile is limited. The presence of Gonystylus species is also critically endangered on the IUCN Red List. Therefore, exploring the -omics profiles of Gonystylus bancanus, a native plant from Borneo Island, is important for Indonesia to conserve the population. This research investigated the metabolite profiling of G. bancanus oil, especially the volatile components of its essential oils. Distillations were performed in two technical ways: hydrodistillation on a laboratory scale and steam distillation on an industrial scale. According to LC–MS and GC–MS profiles, both essential oils displayed similar chemical compositions. This article also discusses the similarity of the chemical contents of gaharu bouya oil and agarwood oil from the gaharu superior type (Aquilaria) to support the value of the oil. This research also investigated the cytotoxicity of gaharu bouya oil against three cell lines: HeLa, MCF-7, and HT-29.

Basal plate rot disease is an important disease of shallots that causes losses in the field and storage. The disease is caused by Fusariumcomplex species, with different levels of virulence and host susceptibility. The six species of Fusariumspp.reported to cause basal plate rot disease of onion were F. oxysporum f.sp. cepae, F. proliferatum, F. redolens, F. solani, F. acutatumand F. tricinctum. Information about the variation in symptoms and morphology of fungi that cause basal plate rot disease on shallots, especially in Indonesia, is still very limited. Besides, the species that cause basal plate rot disease on shallots has not been molecularly confirmed using specific primers. The objective of this study was to isolate, identify, and study the variationin symptoms of the fungus associated with basal plate rot disease. The fungi were isolated from shallot plants symptomatic of basal plate rot disease collected from Brebes, Central Java Province. Eight isolates of Fusariumwith different morphologies weresuccessfully isolated and identified. Five isolates were identified as F. solani (isolate BC1, BC2, BC3, BBS1, and BBS4), two isolates as F. oxysporum f.sp. cepae (isolate BC4 and BBS6) and one isolate F. proliferatum (isolate BBS5). All eight isolates were pathogenic on shallots with different levels of virulence and symptom variations. BC4 isolate had the highest level of virulence, resulting in plant death, and was identified as F. oxysporum f.sp. cepae.

Liriomyza spp.(Diptera: Agromizidae) is a polyphagous pest that attacks various types of vegetable and ornamental plants throughout the world. The most damaging species are Liriomyza huidobrensis(Blanchard), Liriomyza sativae(Blanchard), and Liriomyza trifolii(Burgess). There have been no reports regarding mapping of population distribution and population genetics of Liriomyzain the Bali region using theCytochrome C Oxidase Subunit I (COI) approach. This research aims to map the population and genetic structure of L. huidobrensis, L. sativaeand L. trifoliion vegetable plants in Bali. This research uses a purposive sampling method which represents vegetable plantings in each district and city in Bali Province. Identification of the Liriomyzaspp.gene structure using the COI approach using LCO and HCO primers. The results of the research found that the spatial distribution of invasive leafminer fly species, namely L. sativaeand L. trifolii, was evenly distributed throughout the city districts in Bali. The distribution pattern of L. trifoliiis uniform with an S2/X value <1, while L. huidobrensisis found in the highland areas with a clustered distribution patternwith an S2/.Thegenetic distance between the three Liriomyzaspecies found in vegetable plants in Bali is much longer. This means that the three Liriomyzaspecies show very high levels of genetic differentiation in mitochondrial and nuclear genes, and differentiation between species in nuclear genes. This genetic variation can influence the ability of insects to quickly adapt to new environments and contribute to population dynamics and dispersal capacity.

Lactococcus garvieae is a gram-positive ovoid cocci bacterium formerly classified as a member of the Lactococcus genus. This study aims to isolate L. garvieae from catfish rearing pond and characterize it as a potential probiotic candidate. L. garvieae was identified and characterized through phenotypic and genotypic observation, genomic % G~C content analysis, cell surface hydrophobicity assays, acidification test, in vitro antagonism, and a profile of antimicrobial activities. The MT597595.1 accession number corresponds to L. garvieae, as determined by a molecular identification test. Biochemical characterization was performed using API 50 CH kit. The genomics %G~C content of L. garvieae was 51.8. Findings from acidification ability tests, in vitro antagonism tests, and the ability of bacteria to grow in broth medium at pH 4 reveal that L. garvieae can inhibit the growth of Aeromonas hydrophila, Streptococcus agalactiae, Streptococcus iniae, and Edwardsiella ictaluri. However, it does not suppress the growth of L. garvieae Edwardsiella tarda. Remarkably, L. garvieae has the ability to reduce the pH of neutral broth medium turning it acidic. Furthermore, L. garvieae’s hydrophobic cell surface exhibited an adhesive, hydrophobic,and protein surface cell content with a compact growth pattern consistent with postive SAT and MATH assay. Antimicrobial activity tests, encompassing 11 antibiotics, disclosed resistance to Nalidixic acid while displaying intermediate sensitivity to Streptomycin and Trimethoprim. In conclusion, L. garvieae demonstrates an inhibitory effect on the growth of pathogenic bacteria, underlining its potential as a probiotic candidate.

Rhizobia are bacteria that symbiosis with host plant, as shown in the root nodules formation, and provide nitrogen that can be absorbed by plants in greater quantities than rhizobacteria. Available Nitrogen, which absorbed by plants, is the essential requirement for plant growth because its role in increasing yield and quality, hence it is needed in greater quantities than other nutrients. The study aimed to determine the macroscopic and microscopic diversity of rhizobia isolates from the groundnut nodules and their potential as PGPRs, andto identify 16S rRNA isolates with the best potential as PGPRs molecularly. The methods used were isolation from root nodules, screening of PGPR potential, molecular identification based on the 16S rRNA gene, and phylogenetic analysis to determine their kinship. Based on the isolation results, 17 Gram–negative isolates were obtained white to pink or orange color on AG media with various colony characteristics in terms of shape, margin, elevation, and texture. KT 20, which was selected as rhizobia isolate with the best potential as PGPR, has ammonium concentration of 23.12 ppm, synthesizes IAAwithaconcentration of 10.36 ppm, and phosphatessolubilization activity, although its ability to synthesize proteases is low. The results of molecular identification of 16S rRNA showed that KT 20 belongs to the Rhizobiumgenus witha similarity of 99.48% andbootstrap valueof 96%.

Green macroalgae, known locally as Latoh, is one of the green seaweeds consumed by the local community in Jepara and is beneficial for health. This study explores the potential of secondary metabolites from seaweed and its symbiotic bacteria as natural food preservatives and antibacterials. Seaweed samples were collected from the seagrass ecosystem of Panjang Island, Jepara, Indonesia. Subsequently, the samples were subjected to scanning electron microscopy analysis, proximate analysis, phytochemical analysis, thin layer chromatography, and high–performance liquid chromatography for amino acid analysis. A sample was subjected to a multistage extraction process using n–hexane, ethyl acetate, and methanol (1:5 w/v), each for 24 h. Symbiotic bacteria from seaweed were isolated, and enzymatic (proteolytic, amylolytic, and cellulolytic) and antibacterial testing against pathogenic bacteria Staphylococcus aureusand Escherichia coliwas conducted using the disc diffusion method. The selected bacteria were subjected to molecular identification. The research showed that Caulerpa lentilliferahad an ash content of 3.24%, protein content of 0.57%, and fat content of 0.337%. Phytochemical analysis shows that the sample contains flavonoids, steroids, and alkaloids. HPLC analysis reveals that Caulerpa lentilliferahas the highest content of aspartic acid (relative area: 11.90%), glutamic acid (relative area: 13.43%), and alanine (relative area: 9.03%). Caulerpa racemosasample shows the highest detector response for glutamic acid (relative area: 12.19%), aspartic acid (relative area: 11.10%), and alanine (relative area: 9.63%). The results indicate that 14 bacterial isolates were successfully isolated, with 6isolates from Caulerpa lentilliferaand 8isolates from Caulerpa racemosa,all exhibiting enzymatic and antibacterial abilities. The research results concluded that the Latoh seaweed species Caulerpa lentillifera andCaulerpa racemosaand their symbiotic bacteria have the potential to be used as food preservatives

Trichoderma has the potential to suppress fungal pathogens and thus control plant diseases, including Phytophthora foot rot, which is the most devastating disease of black pepper in Lampung. Identification of a microorganism can not only rely on its morphological characteristics, but it is also necessary to identify it molecularly at the species level. This research was aimed at identifying the fungus Trichoderma sp. Margodadi isolates at the species level and to know the potential of Trichoderma sp. Margodadi isolates and their secondary metabolites to control P. capsici. This research was conducted from March to November 2021 at the Laboratory of Plant Disease, Department of Plant Protection, and the Laboratory of Agricultural Biotechnology, Faculty of Agriculture, University of Lampung. Identification of Trichoderma was done by morphological characteristics and molecular methods. The ability of Trichoderma to suppress P. capsici was tested by dual culture. The effect of secondary metabolites on the growth of P. capsici was determined in vitro at concentrations of 0% (control), 10%, 20%, 30%, and 40%. The experimental design used was a completely randomized design consisting of five treatments repeated five times. The data obtained from the test were analyzed using ANOVA, followed by the LSD test at 5%. The results of this study showed that Trichoderma sp. Margodadi isolate had a close relationship with Trichoderma asperellum and had the ability as an antagonist to inhibit the growth of P. capsici up to 47.23%, and the secondary metabolites produced could inhibit the growth of P. capsici up to 72.53% with the best concentration of 40%. 

Polyethylene terephthalate (PET) plastic is the most widely used type of plastic that produces waste and causes various environmental and health problems. The treatment of PET plastic waste with chemically and mechanically recycling approaches still has shortcomings, so biological processing using microorganisms or enzymes has new potential. Two bacterial isolates from the Indonesian Culture Collection of NationalResearch and Innovation Agency (InaCC, BRIN), namely isolate InaCC B538 and InaCC B947, were further observed for their potential in PET plastic degradation. Firstly, both isolates were determined by the molecular marker 16S rDNA. The potential of both isolates was measured with following method: 10 days of degradation using PET and PCL substrates, esterase enzyme activity assay, and observation of the PET plastic surface using Scanning Electron Microscope (SEM). Species identification was performed using DNA sequencing of 16S rDNA. InaCC B538 and InaCC B947 were closely related to Bacillus altitudinis TBMAX41 and Alcaligenes faecalis AN-13, respectively. InaCC B947 isolate has a better potential in degrading PET plastic and PCL with a degradation percentage of 0.32% for PET plastic and 3.22% for PCL film for 10 days, respectively, and esterase activity of 0.06 U/ml; while InaCC B538 did not cause weight loss of PET and 2.49% for PCL, respectively, with esterase activity of 0.04 U/ml. The degradation of PET plastic by the isolates InaCC B947 was able to cause damage to the plastic surface leading to the degradation of PET plastic.

The Nias Islands are an archipelago in the western part of North Sumatera, encompassed by the Indian Ocean, and have become a hotspot for demersal and pelagic fishes. These geographical conditions endow the waters around Nias with large fishery resources, leading to their frequent utilization as fishing grounds for Sumatera Island and its surrounding areas. This research aimed to identify the commercially important fish species with the highest catch frequency in the waters of Nias, and this identification marked the initial stage of the sustainable utilization of fish resources. The method used was DNA Barcoding using genes targeting the COI Mitochondrial locus. Therefore, 43samples were collected from several North and South Nias fish landing sites. The obtained species were classified into 3 groups based on the commodity type: demersal fish, pelagic fish, and Choncricthyes. There were 8 species of demersal fish (Caesio caerulaurea, Halichoeres scapularis, Lethrinus ornatus, Mulloidichthys flavolineatus, Parupeneus barberinus, Scarus prasiognathos, Plectropomus leopardus, andVariola albimarginata), displaying genetic distances ranging from 0.002–0.275. Pelagic fish consisted of 5 species, namely Amblygaster clupoides, Caranx ignobilis, Caranx sexfasciatus, Ferdauia ferdau, andScomberomorus commerson, displaying genetic distances ranging from 0.002–0.267. The next commodities were Chondrichthyeswith 9 species (Carcharhinus sealei, Himantura leoparda, Neotrygon kuhlii, Paragaleus randalli, Pateobatis jenkinsii, Rhynchobatuscf laevis, Spyrna lewini, Taeniura meyeni, andUrogymnus granulatus), displaying genetic distance ranging from 0–0.271.This value indicates that migratory species such as Chondrichthyeshave quite extensive movements so that the genetic distance between species and between populations tends to be low.

Pemphis acidulais a wild plant in rocky or sandy coastal areas and mangrove ecosystems. Different geographic characteristics may affect plant adaptability and have an impact on the emergence of various genotypes. This study was performed to reveal the phenetic relationship and genetic variation of P. acidu-lain 3 different areas in Tomini Bay, Gorontalo Province, Indonesia. We took 3 samples from each location and analysed them using 14 morphological char-acters and molecular approaches based on ISSR markers and ITS gene. The results showed that P. acidulaon Olele had bigger sizes in some morphological features compared to the plants in other study areas. The phenetic analysis showed that P. acidulaat Biluhu and Dulanga were more closely related, alt-hough P. acidulaat the 3 locations had 100% similarity. Genetic variation anal-ysis showed the highest genetic similarity based on ISSR markers was found in Dulanga and Biluhu samples (76.8%). Phylogenetic based on ITS gene re-vealed that Olele samples were in the same clade with P. acidula accession from GenBank (genetic distance 0-0.19%), while Biluhu samples were a sister group (genetic distance 24.97-25.03%) even though their percentage identity corre-sponds to P. acidula (81.34%). Plant adaptation to different habitat conditions may affect the genetic diversity of P. acidula.

Background and Aims: Despite the endemicity of Japanese encephalitis virus (JEV) in humans and animals in the Province of Bali, Indonesia, there is little data on whether seroconversion to the virus occurs in pigs, JEV genotypes circulating, and it’s potential mosquito vectors in the area. The aims of this study were to (i) Determine whether JEV infection in Balinese pigs occurs before reaching their sexual maturity, (ii) identify the genotypes of circulating JEV, and (iii) identify potential JEV mosquito vectors at the study sites in urban and peri-urban areas of Bali. Materials and Methods: Sixteen 1-week-old Landrace piglets from two different sows were housed in Denpasar. Similarly, 18 one-week-old mixed-breed piglets of two different sows were housed in Badung Regency. The piglets were bled every 1 to 4 weeks for up to 24 weeks. Serum samples from the 11 piglets were tested for antibodies against JEV, and seroconversion- suspected sera were titrated using an enzyme-linked immunosorbent assay. Blood of seroconverted sera from pigs were tested using polymerase chain reaction (PCR) to detect the genetic sequence of JEV. The mosquitoes in the sentinels were trapped throughout the study period to identify the potential mosquito vectors of JEV. Results: Antibodies were detected in most of the selected piglets’ sera from weeks 1 to 24 of their age. However, sera of pig B9 collected from the sentinel setting in Badung Regency showed a four-fold increase in antibody titer from week 4 to week 8, indicating seroconversion. PCR testing of blood from B9 (pooled blood sample collected from week 5 to week 8) identified JEV nucleic acids, which were phylogenetically classified as belonging to the JEV genotype III. Meanwhile, 1271 of two genera of mosquitoes, Anopheles spp. and Culex spp. were trapped in the pig sentinels. Conclusion: JEV seroconversion likely occurs before the pig reaches sexual maturity in Badung Regency. Sequence data indicate that JEV genotype III is circulating in the pig sentinel setting in the regency; however, circulating genotypes need to be clarified through increased surveillance. Meanwhile, Culex spp. and most likely Culex quinquefasciatus and Anopheles spp. were the dominant mosquitoes present in the study sites set in the urban area of Denpasar and peri-urban areas of Badung, Bali, indicating that these are likely vectors in spread of JEV in the region

This research aims to quantify antiquorum sensing and antibiofilm activity of f phyllosphere bacteria against biofilm formed by pathogenic fish bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. Antiquorum sensing assay using Chromobacter violaceum as indicator bacteria and antibiofilm assay showed six phyllosphere bacteria have antiquorum sensing and antibiofilm activities against tested bacteria. The highest inhibition and destruction activity was showed by metabolite of JB 3B and EJB 5 F against A. hydrophila, respectively. Determination using light microscope and scanning electron microscope performed decreaing in biomass of biofilm observed after treated with metabolite from phyllosphere bacteria.