Promoter regulates expression level of foreign gene in transgenic organism. This study was perfor... more Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select a suitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Green fl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycus alvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP), medaka β-actin (pmBA-GFP) and Japanese fl ounder keratin (pJfKer-GFP) promoters were introduced by electroporation method. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms, pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expression level using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus (34.10%±1.49) expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activity with CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expression at medium level and similar number of fi lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoter had lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCR analysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressing fi lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters was an appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combining this achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K. alvarezii can be feasible.
Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be use... more Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR ...
Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the “new normal” peri... more Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the “new normal” period, where people’s activity has bounced back, followed by the easing of travel policy restrictions. The lesson learned is that the wide availability of accurate and rapid testing procedures is crucial to overcome possible outbreaks in the future. Therefore, many laboratories worldwide have been racing to develop a new point-of-care diagnostic test. To aid continuous innovation, we developed a plasmonic-based biosensor designed explicitly for portable Surface Plasmon Resonance (SPR). In this study, we designed a single chain variable fragment (scFv) from the CR3022 antibody with a particular linker that inserted a cysteine residue at the second position. It caused the linker to have a strong affinity to the gold surface through thiol-coupling and possibly become a ready-to-use bioreceptor toward a portable SPR gold chip without purification steps. The theoretical affinity of this scFv on ...
Dengue virus serotype 3 has been recognized as the predominant serotype in many occurrences of De... more Dengue virus serotype 3 has been recognized as the predominant serotype in many occurrences of Dengue Hemorrhagic Fever (DHF) in Indonesia. Performing DHF prevention could be done with vaccination. BPPT institution was developing a vaccine, which is made from NS2B-NS3 recombinant protein. This protein is one of the non-structural proteins that arrange the genome of DENV and it has a molecular weight of 83 kDa. This research aims to isolate and purify NS2B-NS3 protein DENV-3 from transformant cell of Saccharomyces cerevisiae. Purification of NS2B-NS3 protein is done with HisPur Ni-NTA Magnetic Beads method. Optimization of purification is done by increasing the concentration of imidazole as protein binder in the elution buffer starting from 250 mM until 500 mM. The validity of purified protein was tested qualitatively with Sodium Dodecyl Sulfate-Polyacrilamide Gel Electrophoresis (SDS-PAGE) method and quantified by Bichinconinic Acid (BCA) method. The results revealed us that NS2B-NS...
Humpback grouper is one of the most cultured fishes in Asia, including Indonesia. The main proble... more Humpback grouper is one of the most cultured fishes in Asia, including Indonesia. The main problem faced by humpback culture is its slow growth rate. One of the methods that will be more effective and efficient to solve the problem is using transgenic technique. This study was conducted to determine the effectiveness of transfection, microinjection and electroporation techniques on gene transfer in humpback grouper. transfection was performed by incubating sperm to the foreign DNA (pktBP-ktGH gene construct)-transfectant complex solution, while was by injecting those complex solution into testis of mature males. Microinjection was conducted in 2-4 cell stage embryos using 25 μg/ml of foreign DNA solution, and duration of injection was 1, 2 and 3 seconds. Electroporation by 50 V, 30 ms of pulse length, 5 of pulse number and 0.1 of pulse interval was performed to sperm using three DNA concentration of 5, 10 and 20 μg/ml. The incorporation of foreign DNA in sperm and embryos were analy...
Isolation and characterization of B-actin promoter from humpback grouper (Cromileptes altivelis).... more Isolation and characterization of B-actin promoter from humpback grouper (Cromileptes altivelis). By: Alimuddin, Wedaraningtyas Nugrahani, Ratu Siti Aliah, Komar Sumantadinata, Irvan Faizal, Odang Carman, and Goro
Pseudomonas putida strain T-57, which was isolated from activated sludge at a wastewater treatmen... more Pseudomonas putida strain T-57, which was isolated from activated sludge at a wastewater treatment facility of a fermentation plant in Kumamoto, Japan, is a solvent-tolerant aerobic bacterium3). It can grow even when organic solvents such as toluene, ethylbenzene, xylenes, styrene, n-octane, and n-decane are provided in a two-phase (organic-aqueous) system. This bacterium utilizes toluene, p-xylene, ethylbenzene, and n-butanol as sole sources of carbon and energy. Genetic and biochemical analysis revealed that P. putida strain T-57 catabolizes aromatic hydrocarbons via the toluene dioxygenase (TDO) pathway (Fig. 1). From these characteristics, P. putida strain T-57 seems very promising as a biocatalyst for the bioproduction of value added chemicals from hydrophobic substrates. In the previous studies, we investigated the bioproduction of cresol from toluene by using a mutant derived from P. putida T-573,20). In the initial step of the TDO pathway, toluene is oxidized to toluene cis-...
The objective of this study was to compare the growth response of 3 different sizes of humpback g... more The objective of this study was to compare the growth response of 3 different sizes of humpback grouper juveniles, which were come from the same brood stock group and spawning season after “stop and go” experiment of recombinant Epinephelus lanceolatus growth hormone (rElGH); that are with, without and retreated with rElGH for 42 days respectively. Each size group was divided into two treatment groups. The first group was treated with 50 mg crude rElGH kg-1 in commercial diet (pC) and a second group as a control. Weight gain of pC compares to control for small size group, medium size group and large size groups of juveniles subsequently for first stage were 85.89%, 39.66% and 16.34%; second stage were -34.57%, -14.76%, and -5.27%; and third stage were 56.16%, 50.24% and 59.14%. Specific growth rate differences of small, medium and large size of pC compared to control in first stage were 41.6%, 19.06% and 7.52%; second stage were -44.81%, -27.23% and -14.66%; and third stage were 55....
Faizal I, Emdi A. 2017. Analysis of flowering gene in palm oil (Elaeis guineensis). Asian J Agric... more Faizal I, Emdi A. 2017. Analysis of flowering gene in palm oil (Elaeis guineensis). Asian J Agric 1: 53-58. Palm oil has always been an important commodity in Indonesia. The most common species is palm oil, Elaeis guineensis. Palm oil is a monoecious plant with a tendency to be a temporal dioecious. Female flower will be the one that produces palm oil fruit, that later is treated with palm oil while male flower only takes part in the fertilization process. In order to know the ratio between female and male flower tree in a plantation, this study was performed to detect a distinction between female and male flowering gene sequences from DNA sample of E. guineensis. Based on previous study which managed to characterize MADS-box gene of palm oil, a primer was designed and named GmG (Globosa-male-Gaps). The result shows that the primer has the ability to differentiate DNA sequence female and male flower of E.guineensis, Palm oil. However, further studies with full sequence and more samp...
Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasila... more Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasilan transgenesis. Penelitian isolasi dan karakterisasi promoter β-actin (ktBA) dari ikan kerapu bebek (Cromileptes altivelis) dalam rangka pembuatan ikan kerapu autotransgenik telah dilakukan. Promoter β-actin memiliki aktivitas tinggi pada jaringan otot. Sekuens promoter ktBA diisolasi menggunakan metode degenerate PCR. Sekuensing dilakukan menggunakan mesin ABI PRISM 3100. Analisis sekuens menggunakan software BLAST, GENETYX versi 7 dan TFBind. Fragment DNA hasil amplifikasi PCR yang dipotong dari vektor kloning selanjutnya diligasi dengan pEGFPN1 untuk membuat konstruksi pktBA-EGFP. Konstruksi pktBA-EGFP dimikroinjeksi ke embrio ikan zebra (Danio rerio) fase 1 sel untuk menguji aktivitas promoter ktBA. Ekspresi gen EGFP diamati menggunakan mikroskop fluoresens. Analisis sekuens menunjukkan bahwa panjang fragmen DNA hasil amplifikasi PCR sekitar 1,6 kb dan memiliki faktor transkripsi ya...
A toluene-metabolizing bacterium, designated Pseudomonas putida T-57, was isolated from soil samp... more A toluene-metabolizing bacterium, designated Pseudomonas putida T-57, was isolated from soil samples. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10&ndash
The objective of this study was to compare the growth response of 3 different sizes of humpback g... more The objective of this study was to compare the growth response of 3 different sizes of humpback grouper juveniles, which were come from the same brood stock group and spawning season after “stop and go” experiment of recombinant Epinephelus lanceolatus growth hormone (rElGH); that are with, without and retreated with rElGH for 42 days respectively. Each size group was divided into two treatment groups. The first group was treated with 50 mg crude rElGH kg in commercial diet (pC) and a second group as a control. Weight gain of pC compares to control for small size group, medium size group and large size groups of juveniles subsequently for first stage were 85.89%, 39.66% and 16.34%; second stage were -34.57%, -14.76%, and -5.27%; and third stage were 56.16%, 50.24% and 59.14%. Specific growth rate differences of small, medium and large size of pC compared to control in first stage were 41.6%, 19.06% and 7.52%; second stage were -44.81%, -27.23% and -14.66%; and third stage were 55.9%...
Promoter regulates expression level of foreign gene in transgenic organism. This study was perfor... more Promoter regulates expression level of foreign gene in transgenic organism. This study was performed to select a suitable promoter as the fi rst step towards production of valuable trait-enhanced seaweed by transgenic technology. Green fl uorescent protein (GFP) gene was used as a reporter to determine the activity of promoter in seaweed Kappaphycus alvarezii. GFP gene constructs driven by cytomegalovirus (pCMV-GFP), caulifl ower mosaic virus (pCaMV-GFP), medaka β-actin (pmBA-GFP) and Japanese fl ounder keratin (pJfKer-GFP) promoters were introduced by electroporation method. Electroporation was performed using a gene pulser (BIORAD) with voltage of 300 V, pulse length of 0.5 ms, pulse numbers of 4, and pulse interval of 0.1 s. Promoter activity was determined by analyzing GFP gene expression level using a fl uorescent microscope. The results showed that CMV regulated highest number of fi lament callus (34.10%±1.49) expressing GFP at medium to strong fl uorescence levels. CaMV promoter had relatively similar activity with CMV, but lower number of fi lament callus expressing GFP (10.48%±0.25). mBA promoter drove GFP expression at medium level and similar number of fi lament callus (8.85%±2.31) expressing GFP with CaMV, while JfKer promoter had lowest activity by means in number of fi lament callus expressing GFP (4.79%±0.26) and GFP expression level. PCR analysis for transgenic confi rmation showed a DNA band of PCR product from pCMV-GFP and pCaMV-GFP expressing fi lament callus in the same size (about 0.6 kb) with positive control of plasmid. Thus, CMV and CaMV promoters was an appropriate promoter and foreign gene could be transferred to fi lament callus by electroporation method. Combining this achievement with developing a culture method of fi lament callus to be thallus, stable transgenic breeding in K. alvarezii can be feasible.
Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be use... more Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR ...
Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the “new normal” peri... more Two years after SARS-CoV-2 caused the first case of COVID-19, we are now in the “new normal” period, where people’s activity has bounced back, followed by the easing of travel policy restrictions. The lesson learned is that the wide availability of accurate and rapid testing procedures is crucial to overcome possible outbreaks in the future. Therefore, many laboratories worldwide have been racing to develop a new point-of-care diagnostic test. To aid continuous innovation, we developed a plasmonic-based biosensor designed explicitly for portable Surface Plasmon Resonance (SPR). In this study, we designed a single chain variable fragment (scFv) from the CR3022 antibody with a particular linker that inserted a cysteine residue at the second position. It caused the linker to have a strong affinity to the gold surface through thiol-coupling and possibly become a ready-to-use bioreceptor toward a portable SPR gold chip without purification steps. The theoretical affinity of this scFv on ...
Dengue virus serotype 3 has been recognized as the predominant serotype in many occurrences of De... more Dengue virus serotype 3 has been recognized as the predominant serotype in many occurrences of Dengue Hemorrhagic Fever (DHF) in Indonesia. Performing DHF prevention could be done with vaccination. BPPT institution was developing a vaccine, which is made from NS2B-NS3 recombinant protein. This protein is one of the non-structural proteins that arrange the genome of DENV and it has a molecular weight of 83 kDa. This research aims to isolate and purify NS2B-NS3 protein DENV-3 from transformant cell of Saccharomyces cerevisiae. Purification of NS2B-NS3 protein is done with HisPur Ni-NTA Magnetic Beads method. Optimization of purification is done by increasing the concentration of imidazole as protein binder in the elution buffer starting from 250 mM until 500 mM. The validity of purified protein was tested qualitatively with Sodium Dodecyl Sulfate-Polyacrilamide Gel Electrophoresis (SDS-PAGE) method and quantified by Bichinconinic Acid (BCA) method. The results revealed us that NS2B-NS...
Humpback grouper is one of the most cultured fishes in Asia, including Indonesia. The main proble... more Humpback grouper is one of the most cultured fishes in Asia, including Indonesia. The main problem faced by humpback culture is its slow growth rate. One of the methods that will be more effective and efficient to solve the problem is using transgenic technique. This study was conducted to determine the effectiveness of transfection, microinjection and electroporation techniques on gene transfer in humpback grouper. transfection was performed by incubating sperm to the foreign DNA (pktBP-ktGH gene construct)-transfectant complex solution, while was by injecting those complex solution into testis of mature males. Microinjection was conducted in 2-4 cell stage embryos using 25 μg/ml of foreign DNA solution, and duration of injection was 1, 2 and 3 seconds. Electroporation by 50 V, 30 ms of pulse length, 5 of pulse number and 0.1 of pulse interval was performed to sperm using three DNA concentration of 5, 10 and 20 μg/ml. The incorporation of foreign DNA in sperm and embryos were analy...
Isolation and characterization of B-actin promoter from humpback grouper (Cromileptes altivelis).... more Isolation and characterization of B-actin promoter from humpback grouper (Cromileptes altivelis). By: Alimuddin, Wedaraningtyas Nugrahani, Ratu Siti Aliah, Komar Sumantadinata, Irvan Faizal, Odang Carman, and Goro
Pseudomonas putida strain T-57, which was isolated from activated sludge at a wastewater treatmen... more Pseudomonas putida strain T-57, which was isolated from activated sludge at a wastewater treatment facility of a fermentation plant in Kumamoto, Japan, is a solvent-tolerant aerobic bacterium3). It can grow even when organic solvents such as toluene, ethylbenzene, xylenes, styrene, n-octane, and n-decane are provided in a two-phase (organic-aqueous) system. This bacterium utilizes toluene, p-xylene, ethylbenzene, and n-butanol as sole sources of carbon and energy. Genetic and biochemical analysis revealed that P. putida strain T-57 catabolizes aromatic hydrocarbons via the toluene dioxygenase (TDO) pathway (Fig. 1). From these characteristics, P. putida strain T-57 seems very promising as a biocatalyst for the bioproduction of value added chemicals from hydrophobic substrates. In the previous studies, we investigated the bioproduction of cresol from toluene by using a mutant derived from P. putida T-573,20). In the initial step of the TDO pathway, toluene is oxidized to toluene cis-...
The objective of this study was to compare the growth response of 3 different sizes of humpback g... more The objective of this study was to compare the growth response of 3 different sizes of humpback grouper juveniles, which were come from the same brood stock group and spawning season after “stop and go” experiment of recombinant Epinephelus lanceolatus growth hormone (rElGH); that are with, without and retreated with rElGH for 42 days respectively. Each size group was divided into two treatment groups. The first group was treated with 50 mg crude rElGH kg-1 in commercial diet (pC) and a second group as a control. Weight gain of pC compares to control for small size group, medium size group and large size groups of juveniles subsequently for first stage were 85.89%, 39.66% and 16.34%; second stage were -34.57%, -14.76%, and -5.27%; and third stage were 56.16%, 50.24% and 59.14%. Specific growth rate differences of small, medium and large size of pC compared to control in first stage were 41.6%, 19.06% and 7.52%; second stage were -44.81%, -27.23% and -14.66%; and third stage were 55....
Faizal I, Emdi A. 2017. Analysis of flowering gene in palm oil (Elaeis guineensis). Asian J Agric... more Faizal I, Emdi A. 2017. Analysis of flowering gene in palm oil (Elaeis guineensis). Asian J Agric 1: 53-58. Palm oil has always been an important commodity in Indonesia. The most common species is palm oil, Elaeis guineensis. Palm oil is a monoecious plant with a tendency to be a temporal dioecious. Female flower will be the one that produces palm oil fruit, that later is treated with palm oil while male flower only takes part in the fertilization process. In order to know the ratio between female and male flower tree in a plantation, this study was performed to detect a distinction between female and male flowering gene sequences from DNA sample of E. guineensis. Based on previous study which managed to characterize MADS-box gene of palm oil, a primer was designed and named GmG (Globosa-male-Gaps). The result shows that the primer has the ability to differentiate DNA sequence female and male flower of E.guineensis, Palm oil. However, further studies with full sequence and more samp...
Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasila... more Promoter sebagai regulator ekspresi gen merupakan salah satu faktor yang mempengaruhi keberhasilan transgenesis. Penelitian isolasi dan karakterisasi promoter β-actin (ktBA) dari ikan kerapu bebek (Cromileptes altivelis) dalam rangka pembuatan ikan kerapu autotransgenik telah dilakukan. Promoter β-actin memiliki aktivitas tinggi pada jaringan otot. Sekuens promoter ktBA diisolasi menggunakan metode degenerate PCR. Sekuensing dilakukan menggunakan mesin ABI PRISM 3100. Analisis sekuens menggunakan software BLAST, GENETYX versi 7 dan TFBind. Fragment DNA hasil amplifikasi PCR yang dipotong dari vektor kloning selanjutnya diligasi dengan pEGFPN1 untuk membuat konstruksi pktBA-EGFP. Konstruksi pktBA-EGFP dimikroinjeksi ke embrio ikan zebra (Danio rerio) fase 1 sel untuk menguji aktivitas promoter ktBA. Ekspresi gen EGFP diamati menggunakan mikroskop fluoresens. Analisis sekuens menunjukkan bahwa panjang fragmen DNA hasil amplifikasi PCR sekitar 1,6 kb dan memiliki faktor transkripsi ya...
A toluene-metabolizing bacterium, designated Pseudomonas putida T-57, was isolated from soil samp... more A toluene-metabolizing bacterium, designated Pseudomonas putida T-57, was isolated from soil samples. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10&ndash
The objective of this study was to compare the growth response of 3 different sizes of humpback g... more The objective of this study was to compare the growth response of 3 different sizes of humpback grouper juveniles, which were come from the same brood stock group and spawning season after “stop and go” experiment of recombinant Epinephelus lanceolatus growth hormone (rElGH); that are with, without and retreated with rElGH for 42 days respectively. Each size group was divided into two treatment groups. The first group was treated with 50 mg crude rElGH kg in commercial diet (pC) and a second group as a control. Weight gain of pC compares to control for small size group, medium size group and large size groups of juveniles subsequently for first stage were 85.89%, 39.66% and 16.34%; second stage were -34.57%, -14.76%, and -5.27%; and third stage were 56.16%, 50.24% and 59.14%. Specific growth rate differences of small, medium and large size of pC compared to control in first stage were 41.6%, 19.06% and 7.52%; second stage were -44.81%, -27.23% and -14.66%; and third stage were 55.9%...
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