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Background: Background: Ursodeoxycholic acid (UDCA), a secondary bile acid, is an acidic steroid synthesized from cholesterol in hepatocytes. UDCA is widely used for the treatment of various diseases related to liver injury. The use of UDCA to treat non-liver diseases has also been developed recently, such as neurodegenerative diseases, cancer, and obesity. Due to the important role of UDCA on human health, numerous studies in understanding its chemical and pharmacological properties have been published. Methods: Literature sources were obtained from online databases such as Science Direct, Google Scholar, Scopus and PubMed using keywords relating to the purpose of study. Critical analysis and review were performed for all literatures. Results: UDCA is a steroid compound with pharmacological properties. Seventeen enzymes are involved in its biosynthesis, which has been proposed in four pathways: classic, alternative, the Yamazaki, and 25-hydroxylation pathways. UDCA can be isolated from bovine bile, bear bile or all Ursidae, human, rabbit, cow, rat, hamster, sheep, pig, and plant. UDCA has been used in the treatment of several diseases such as primary biliary cirrhosis, intrahepatic cholestasis of pregnancy, hepatolithiasis associated with Caroli syndrome gallstones, cystic fibrosis, hepatitis C virus, chronic heart failure, neurodegenerative diseases, and obesity, as well as in the prevention of cancer. Conclusion: UDCA has a wide range of the pharmacological properties. Further investigations on its efficacy and safety on humans are required before it could be used for several indications. All genes which are responsible for UDCA biosynthesis have been elucidated. That said, further genetic engineering studies in order to find other prospective sources of UDCA could be a challenge for the future research.

The promoter is a part of the gene that functions in carrying out the gene expression, and its work activity becomes a matter of concern to ensure that expression works effectively. MeEF1A6 (Manihot esculenta Elongation Factor 1 Alfa - 6) is a promoter derived from cassava plants (Manihot esculenta). In previous studies, the MeEF1A6 promoter was successfully isolated, introduced, and characterized into the pBI121 plasmid, replacing the CaMV35S promoter. This study aims to analyze the activity of MeEF1A6 promoters in-vivo and in-vitro by using transient and transgenic techniques in tobacco plants. The pBI121 plasmid containing the MeEF1A6 promoter was introduced into Agrobacterium tumefaciens strain AGL1 and LBA4404. The promoter's work was then analyzed by the result of introducing it into the tobacco plant using the transient and stable transformation. The whole part of explants was used for transient study and tested in a minimum of two biological replicates. Sixty sheets of explant leaves that have been cut were used for stable transformation. The promoter work analysis was carried out with the GUS gene expression that integrated with the promoter with histochemical GUS assay. The transient produced a blue color in the roots, stems, and leaves on the whole repetition. The transverse incision in the stem shows the blue color on the epidermis and procambium tissue. Stable transformation using AGL1 as vector produced 43 shoots from 40 calli. A total of 43 shoots were selected with antibiotics and produced 27 plantlets that were successfully grown. Some plantlets are then reacted with x-gluc as histochemical GUS assay substrat and produced a blue color in the explants, indicating that the MeEF1A6 promoter has been successfully introduced. The results indicate that the MeEF1A6 promoter could work on plant tissue in roots, stems, leaves, and tissues that connect meristems such as procambium in tobacco plants. This reinforces the suspicion that the MeEF1A6 promoter performs work constitutionally as a constitutive promoter.

Surfactants are amphiphilic molecules, which have hydrophilic and hydrophobic groups. Surfactants have an important role in various fields including agriculture, cosmetics, pharmaceuticals, bioremediation, and the petroleum industry especially EOR but because synthetic surfactants are not biodegradable, it is necessary to produce biodegradable surfactants such as rhamnolipid biosurfactants. Rhamnolipid is a glycolipid biosurfactant produced by Pseudomonas aeuruginosa. This species is a pathogen, so it is needed to overcome this problem by cloning the rhamnolipid gene into Escherichia coli for large-scale production. Rhamnolipid biosynthesis includes three main genes, rhlA, rhlB, and rhlC. The rhlAB produces mono-rhamnolipid and rhlABC produces di-rhamnolipid. The construction involved one plasmid pPM RHLABC (di-rhamnolipid) with T7lac promoter. Characterization of surfactants by E24, IFT, and CMC analysis showed that di-rhamnolipid biosurfactant has the best activity (70%, 0.8 mN/m, and 300 mg/L) than chemical surfactant, sodium dodecyl sulfate (46%, 4.7 mN/m, and 2000 mg/L) at pH 7, 25 °C, and 0% salinity. The conclusion from this research shows that the characteristics of di-rhamnolipid are very promising in the utilization of industrial-scale including EOR technology, agriculture, and pharmacy

Reducing oxidative species to non- or less-reactive matter is the principal function of an antioxidant. Plant-based food is the main external source of antioxidants that helps protect our cells from oxidative damage. During postharvest storage and distribution, fruits and vegetables often increase ROS production that is quenched by depleting their antioxidant pools to protect their cells, which may leave none for humans. ROS are molecules produced from oxygen metabolism; some of the most widely analyzed ROS in plants are singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. ROS concentration and lifetime are determined by the availability and composition of the antioxidant system that includes enzymatic components such as SOD, CAT, and APX and nonenzymatic components such as vitamins, polyphenols, and carotenoid. Depending on its concentration in the cell, ROS can either be harmful or beneficial. At high concentrations, ROS can damage various kinds of biomolecules such as lipids, proteins, DNA, and RNA, whereas at low or moderate concentrations, ROS can act as second messengers in the intracellular signaling cascade that mediates various plant responses. Novel postharvest methods are sought to maintain fruit and vegetable quality, including minimizing ROS while preserving their antioxidant content.

Background: Immediate and accurate diagnosis of malaria is essential for effective control of this disease. Immunochromatographic based rapid diagnostic tests (RDTs) are economical, simple to perform, and provide results in a relative short time, can be useful to assist effective management of malaria. The commercially available malaria RDT in Indonesia is still imported. Therefore, an effort to produce malaria RDT independently is necessary. One of the biomarkers used in RDTs is Plasmodium lactate dehydrogenase pLDH. The production and accumulation of pLDH during asexual stage or blood-stage in all human infected malaria parasites can be used to indicate parasites viability, which is correlated with the number of parasites present in the plasma of infected patients. Objective: The aim of this research is to produce recombinant PfLDH in Escherichia coli BL21(DE3). Methods: PfLDH gene was cloned into pET30a expression vector to obtain a 6.2 kbp recombinant plasmid pET30a-PfLDH. E. coli BL21(DE3) was transformed with pET30a-PfLDH using the heat shock method. Then, E. coli BL21(DE3)- pET30a-PfLDH was cultured in LB broth containing 50 mg/mL kanamycin and was induced by 1mM IPTG at 37oC. Results: SDS-PAGE and Western Blot analysis showed that recombinant PfLDH was expressed with molecular mass ~30 kDa. Conclusion: Recombinant PfLDH is expressed in E. coli BL21(DE3) and can be used in further research for producing rPfLDH as a biomarker for malaria RDT development.

Introduction. A halophilic bacterium of theHalomonas elongataBK-AG25 has successfully produced ectoine with high productivity. To overcome the drawbacks of high levels of salt in the production process, a nonhalophilic bacteria ofEscherichia coli(E. coli) was used to express the ectoine gene cluster of the halophilic bacteria, and the production of ectoine by the recombinant cell was optimized.Methods. The ectoine gene cluster from the halophilic bacterium was isolated and inserted into an expression plasmid of pET30(a) and subsequently transformed intoE. coliBL21 (DE3). Production of ectoine from the recombinantE. coliwas investigated and then maximized by optimizing the level of nutrients in the medium, as well as the bioprocess conditions using response surface methodology. The experimental designs were performed using a central composite design.Results. The recombinantE. colisuccessfully expressed the ectoine gene cluster ofHalomonas elongataBK-AG25 under the control of theT7promoter. The recombinant cell was able to produce ectoine, of which most were excreted into the medium. The optimization of ectoine production with the response surface methodology showed that the level of salt in the medium, the incubation temperature, the optical density of the bacteria before induction, and the final concentration of the inducer gave a significant effect on ectoine production by the recombinantE. coli. Interestingly, the level of salt in the medium and the incubation temperature showed an inverse effect on the production of intracellular and extracellular ectoine by the recombinant cell. At the optimum conditions, the production yield was about 418 mg ectoine/g cdw (cell dry weight) after 12 hours of incubation.Conclusion. This study is the first report on the expression of an ectoine gene cluster ofHalomonas elongataBK-AG25 inE. coliBL21, under the control of theT7promoter. Optimization of the level of nutrients in the medium, as well as the bioprocess condition using response surface methodology, has successfully increased the production of ectoine by the recombinant bacteria.

Rhamnolipid is a potent biodegradable surfactant, which frequently used in pharmaceutical and environmental industries, such as enhanced oil recovery and bioremediation. This study aims to engineer Escherichia coli for the heterologous host production of rhamnolipid, to characterize the rhamnolipid product, and to optimize the production using autoinduction medium and POME (palm oil mill effluent). The construction of genes involved in rhamnolipid biosynthesis was designed in two plasmids, pPM RHLAB (mono-rhamnolipid production plasmid) and pPM RHLABC (di-rhamnolipid production plasmid). The characterization of rhamnolipid congeners and activity using high-resolution mass spectrometry (HRMS) and critical micelle concentration (CMC). In order to estimate rhamnolipid yield, an oil spreading test was performed. HRMS and CMC result show E. coli pPM RHLAB mainly produced mono-rhamnolipid (Rha-C14:2) with 900 mg/L and 35.4 mN/m of CMC and surface tension value, whereas E. coli pPM RHLABC mainly produced di-rhamnolipid (Rha-Rha-C10) with 300 mg/L and 34.3 mN/m of CMC and surface tension value, respectively. The optimum condition to produce rhamnolipid was at 20 h cultivation time, 37 oC, and pH 7. In this condition, the maximum rhamnolipid yield of 1245.68 mg/L using autoinduction medium and 318.42 mg/L using 20% (v/v) of POME. In conclusion, the characteristics of the rhamnolipid by recombinant E. coli is very promising to be used in industries as the most economical way of producing rhamnolipid.

Background: Human papillomavirus (HPV)-45 genotype circulates in high percentage in Bandung area - Indonesia, after HPV-16 and HPV-18. The aim of this study was to analyse variations of major capsid (L1) HPV-45 and its phylogeny. Furthermore in silico protein structure and epitope prediction was explored. Methods: L1 gene of HPV-45 was amplified, sequenced and aligned. Phylogenetic tree had been built and compared with a complete L1 HPV-45 sequence. Structure and epitope prediction of L1 protein were then developed in silico. Results: Of 5 L1 HPV-45 sequences collected, we have detected one variant of sub lineage A2 which was considered as a new variant, and two variants of B2. Superimposition of structure of these two variants with reference showed very similar structure. Furthermore, seven amino acid substitutions were found within these L1 variants of which two substitutions might change the polarity of corresponding amino acid I329T and S383G. The S383G occurred in surface loop (HI-Loop) of new L1 HPV-45 variant. Conclusion: Similar structure of Indonesian variants indicates that amino acids variations do not affect the L1 structure. However, one substitution with altered amino acid polarity found within the area of surface loop suggests a potential impact in antibody recognition and neutralization.

Ectoine is one of the compatible organic molecules that can protect the protein from heating, freezing, and chemicals contact. This study aims to investigate the ability of ectoine to stabilize lipase on heating and in methanol treatments as an effort to provide a stable biocatalyst for the production of biodiesel. Various ectoine concentrations were added to lipase solutions, then the mixture was heated, and the residual activity of the lipase was determined. Similar steps were also conducted for methanol treatment. The results showed that ectoine maintained and even improved the catalytic activity of lipase after treatment with either heat or methanol. The addition of ectoine to a final concentration of 110 to 150 mM could maintain lipase activity up to 80% when heating to approximately 95 °C. Additionally, more than 20% of lipase activity increased on heating to temperatures below 75 °C in the presence of ectoine at a final concentration of 25 to 120 mM. Meanwhile, after incubation in methanol at a level of around 84% (v/v), the activity of lipase containing 40–90 mM ectoine was maintained. These results demonstrated that ectoine was highly effective in protecting lipase from heat and methanol.

Pyrophosphate-dependent Phosphofructokinase or PFP is an enzyme that regulate sucrose metabolism. It consists of α- and β-subunits, which encoded by PFPα and PFPβ genes, respectively. Sugarcane PFPa has a strong function in glycolysis and has the potency to be engineered to increase sugarcane yield. Hence, the purpose of this work was to isolate, clone and characterize the sugarcane PFPα gene. Total RNA was isolated from leafrolls of TD 91 sugarcane variety. cDNA synthesis followed by DNA amplification of PFPα gene were performed using degenerate primers. cDNA and DNA fragments were ligated into pGEM-T Easy vector, which were subsequently introduced to E. coli competent cells. EcoRI were used to cut the plasmids for sequencing. Finally, homology searching was conducted using BLASTn, and then the nucleotide sequence was translated to a protein sequence using Bioedit. The results showed that PFPα cDNA fragment was 900 bp in length. The translated PFPa protein showed binding sites for fructose-6-phosphate and fructose-1,6-biphosphate, which are conserved in all family members of PFP. In silico analysis of the DNA fragment showed gene without intron. In conclusion, the PFPα gene from sugarcane has been successfully isolated, cloned and characterized.

Global demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering-based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens -mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303- dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S-408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co-cultivated on MS co-cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA- dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA- dbr2 construct and transformation into A. annua have been successfully performed.

Surfactant is generally synthetic chemical, which is effective and reliable. However, the chemicals usually did not degraded easily in the environment and could cause damage to the environment. The other possible alternative to produce surfactant is using genetic engineering in order to produce peptide based surfactant. In this research, peptide surfactant was produced using a gene construct which was created using overlapped polymerase chain reaction method (OE-PCR). PAGE analysis shows that single surfactant peptide construction can be expressed by induction of IPTG 1 mM and after at least twice sonication. This research proves that both two constructions have been successfully expressed by producing peptide in expected size (approximately 15 kDa).

Increasing world sugar demand might be fulfilled with land extensification which include the use of dry area. Development of drought tolerance and high productivity sugarcane variety could be achieved by plant genetic engineering. Under drought condition, proline will be accumulated and functioned as an osmoregulator in plant cells. ∆ 1 - pyrroline -5- carboxylate synthase (P5CS) is one of the important enzymes in proline biosynthesis. This enzyme is encoded by P5CS gene family. We cloned two homologous P5CS genes from sugarcane, SoP5CS1 (Accession Number : KF178299) and SoP5CS2 (Accession Number : KF178300), which encode 729 and 716 amino acid polypeptides. The identity between these two genes was 74% based on nucleotide sequences. The SoP5CS1 gene had 98% identity with SbP5CS1 (Accession Number : GQ377719.1) and SoP5CS2 had 99% identity with SaP5CS (Accession Number : EF113257.1). In this experiment, sugarcane plantlets were exposed to medium containing PEG 6000 (40%) for 12, 24, 48, and 72 hours. Proline concentration was measured after treatment and genes expression were analyzed by real time-qPCR. The results showed that the proline concentration was increased 12 folds (9.8 umol.g -1 ) after 48-hours stress treatment. The highest expression of SoP5CS1 occured at 24-hours treatment with approximately 16 times from plant without PEG (control plant) and decreased gradually at 48 and 72 hours treatment. The highest expression of SoP5CS2 occured at 24-hours drought stress with approximately 3.6 folds compared to control. In drought treatment, the expression level SoP5CS1 was higher than SoP5CS2 and has increased significantly at 12-hours treatment. It is suggested that the SoP5CS1 gene contributes more significantly to the production of proline during drought stress than SoP5CS2 . Hence, SoP5CS1 could potentialy be used as a marker to screen sugarcane variety for drought tolerance and for the development of transgenic plant tolerant to drought. Keywords: cloning, drought, expression, P5CS, sugarcane

Artemisinin, a secondary metabolite from Artemisia annua L. is a sesquiterpene lactone that has antimalaria activity but produced at low quantities by the plant. Low levels of artemisinin in the plant is related to the biosynthetic pathways influenced by specific enzymes that play role in the formation of artemisinin. Farnesyl diphosphate (FDP), which is the main precursor of artemisinin, also known as the precursor for the formation of sterols. Compared with the other compounds, sterol biosynthetic pathway is the biggest competitor of the artemisinin production since sterols are needed by plants to regulate membrane fluidity and permeability. This research aimed to study the effect of terbinafin and DMSO as sterol synthesis inhibitors on the regulation of the artemisinin biosynthetic pathway by analyzing the expression level of two genes linked, squalene synthase (SQS) and amorpha-4,11-diene synthase (ADS) using quantitative PCR (qPCR) and the amount of artemisinin is determined using high performance liquid chromatography (HPLC). The results showed that at mRNA levels, terbinafin 30 μM had no significant effect on SQS and ADS expression levels, but it increased the amount of artemisinin at 50 hours incubation up to 1.36 times higher than control. DMSO increased the expression level of ADS up to 3-4 times and increased the content of artemisinin up to 2.42 times higher than control at 50 hours incubation time

Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, palI gene from Klebsiella oxytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length palI gene sequence of Klebsiella oxytoca was cloned in E. coli DH5α. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomerisation 325RLDRD329 and 97% identical to palI gene from Klebsiella sp. LX3 (GenBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNA sequencing result shows that palI gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of palI gene from Klebsiella oxytoca.

Waste from nickel mining of Sorowako in South Sulawesi contains hexavalent chromium, nickel and cobalt metals in high concentration and may have a negative impact to the environment. Common waste treatment systems such as chemical treatment using a reducing reagent may still have a negative impact. Bioremediation using fungi or bacteria becomes more popular because it is an environmentally friendly alternative. The purposes of this study are to isolate and identify indigenous fungi that are resistant to heavy metals (hexavalent chromium, nickel, and cobalt) and are capable of reducing the concentration of metals in mining wastes. Ten fungal isolates were successfully isolated from the soils and pond sediments in the area of nickel mining in Sorowako. Selection of superior isolate was carried out by growing all the isolates on PDA medium, which contained all of the three metals. One superior isolate was identified to be able to grow on medium with concentrations of 6400 ppm hexavalent chromium, 200 ppm nickel and 50 ppm cobalt. Molecular identification and phylogenetic studies of the isolate using fungal PCR primers developed to amplify the ITS (internal transcribed spacer) region showed that the isolate sequence was very close to Trichoderma atroviride with 99.8% similarity. Optimum incubation time for the uptake of hexavalent chromium was 3 days, nickel and cobalt was 5 days, respectively, with an optimum pH of 4.

Leaf colors of a plant can be used to identify stress level due to its adaptation to environmental change. For most leaves green-related colors are sourced from chlorophyll a and b. Chlorophyll concentration is normally measured using a spectrophotometer in laboratory. In some remote observation places, it is impossible to collect the leaves, preserve them, and bring them to laboratory to measure their chlorophyll content. Based on this need, measurement of chlorophyll content is observed through its color. Using CIE chromaticity diagram leaf color information in RGB is transformed into wavelength (in nm). Paddy seed with variety name IR-64 is used in observation during its vegetation stage t (age of 0-10 days). Light exposure time {\tau} is chosen as environmental change, which normally should be about 12 hours/day, is varied (0-12 hours/day). Each day sample from different exposure time is taken, its color is recorded using HP Deskjet 1050 scanner with 1200 dpi, and its chlorophyll content is obtained from absorption spectrum measured using Campspec M501 Single Beam UV/Vis Spectrophotometer after it is rinsed in 85 % acetone solution and the information from the spectrum is calculated using Arnon method. It has been observed that average wavelength of leaf color {\lambda}avg is decreased from 570.55 nm to 566.01 nm as is measured for t = 1 - 10 days with {\tau} = 9 hours/day, but chlorophyll concentration C is increased from 0.015 g/l to 3.250 g/l and from 0.000 g/l to 0.774 g/l for chlorophyll a and b, respectively. Other value of {\tau} gives similar results. Based on these results an empirical relation between concentration of chlorophyll a Cc-a and its wavelength {\lambda}avg can be formulated., Comment: 8 pages, 5 figures, conferene paper to be presented in Padjadjaran International Physics Symposium 2013 (PIPS 2013), 7-8 May 2013, Jatinangor, Indonesia