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All Journal HAYATI Journal of Biosciences The Indonesian Biomedical Journal
Tan, Marselina Irasonia
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Earth & Planetary Sciences Public Health

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Improvement of Plasmid Volumetric Yield by Addition of Glycerol and Phosphate Buffer in Escherichia coli TOP10 Batch Culture Anindyajati; Afifah, Salma Aulia; Riani, Catur; Tan, Marselina Irasonia; Natalia, Dessy; Giri-Rachman, Ernawati Arifin; Artarini, Anita
HAYATI Journal of Biosciences Vol. 31 No. 3 (2024): May 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.3.572-580

Abstract

The investigation of mRNA development has gained substantial interest, particularly in the ex vivo and in vivo therapy. mRNA is widely used for the development of gene editing-based therapies and mRNA vaccines. The aim of this study was to optimize the medium and harvest time to increase plasmid DNA production as part of mRNA production. This study modified used a medium modification approach to achieve high density culture of Escherichia coli TOP10 pGEMT-N in batch cultivation method. Various media formulations were assessed, including LB; LB with phosphate buffer (K2HPO4 12.549 g/L and KH2PO4 2.31 g/L); LB with glycerol (50 g/L); LB with glycerol and phosphate buffer; LB with phosphate buffer, glycerol, glucose (15 g/L), and galactose (15 g/L). The effect of additional carbon sources and phosphate buffer on culture density was measured through OD600 and wet cell weight analysis. The highest OD600 and wet cell weight was observed when LB with glycerol and phosphate buffer was used, with OD600 of 4.78±0.14 and wet cell weight of 36.00±0.63 mg/ml. Plasmid DNA was subsequently isolated from these cultures following 5- and 7.5-hour incubation periods. The utilization of LB medium with glycerol and phosphate buffer resulted in a substantial increase in the volumetric concentration of plasmid DNA of 1,516.97±385.00 ng/ml after 5 hours of incubation. In conclusion, a remarkable enhancement in plasmid DNA volumetric yield within 5 hours was achieved by addition of glycerol and phosphate buffer to LB medium, leading to incubation period.
In Silico Study, Design, and Expression of an Intranasal Dual Chimeric Vaccine for Indonesian-Based Norovirus GII-2 and Hepatitis B Giri-Rachman, Ernawati Arifin; Tan, Marselina Irasonia; Novia Syari Intan; Putri Ayu Fajar; Wojciechowska, Gladys Emmanuella Putri; Hertadi, Rukman; Retnoningrum, Debbie Soefie
HAYATI Journal of Biosciences Vol. 31 No. 5 (2024): September 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.5.1007-1018

Abstract

Hepatitis B virus (HBV) remains an important healthcare challenge, leading to liver diseases like cirrhosis and cancer. In response, we created a prophylactic and therapeutic HBV vaccine by integrating HBcAg and HBsAg from HBV genotype B into Norovirus (NoV) GII.2 P domain (PdomGII.2-HBV) for enhanced intranasal delivery. This vaccine also aimed to simultaneously prevent NoV infection, which causes gastroenteritis. Since the selected HBV epitopes have undergone extensive research and are tailored to the Indonesian population, this study focused on identifying NoV epitopes and assessing T cell epitopes coverage of the PdomGII.2-HBV for the Indonesian population. Following that, we expressed the PdomGII.2-HBV protein using Escherichia coli BL21(DE3) and employed a gentle solubilization technique for protein purification. Our in-silico analysis identified two B cell epitopes, along with 15 CD4+T cell epitopes and 35 CD8+T cell epitopes within the GII.2 P domain. These T cell epitopes cover 100% of the Javanese-Sundanese population's HLA allele variations, which constituted the largest demographic group in Indonesia. Subsequently, we successfully purified the presumed PdomGII.2-HBV protein, revealing a molecular weight of 39.5 kDa. Following the successful expression and purification of the presumed PdomGII.2-HBV protein, it is evident that this vaccine design has significant potential, warranting further study.
In silico–guided Design and Endonuclease-Based Functional Validation of sgRNAs Targeting ERBB2 Transmembrane and Kinase Domains Gunsi, Via Susana; Dirgahyu, Anugrah Prima; Meitha, Karlia; Christianto, Antonius; Tan, Marselina Irasonia
The Indonesian Biomedical Journal Vol 18, No 2 (2026)
Publisher : The Prodia Education and Research Institute (PERI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.18585/inabj.v18i2.4048

Abstract

BACKGROUND: Erythroblastic oncogene B2 (ERBB2) overexpression promotes breast cancer progression through activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase (MAPK) signaling pathways. Although CRISPR–Cas9 enables precise gene disruption, sgRNA efficiency is strongly influenced by target sequence and structural features, necessitating careful in silico design and validation. This study aimed to design and evaluate sgRNAs targeting the transmembrane- and tyrosine kinase–encoding regions of ERBB2.METHODS: Candidate sgRNAs were selected using CHOPCHOP, CRISPOR, and CRISPR RGEN, followed by secondary structure analysis with RNAfold to assess folding stability and scaffold integrity. sgRNAs were synthesized via in vitro transcription and assembled with Cas9 protein to form ribonucleoprotein (RNP) complexes. In vitro endonuclease assays were performed using PCR-amplified ERBB2 fragments derived from SK-OV3 genomic DNA, corresponding to exon 22 (523 bp) and exon 25 (933 bp). sgRNAs targeting eIF4E1 served as positive controls.RESULTS: Two sgRNAs meeting all in silico criteria were selected. Secondary structure prediction confirmed that both sgRNAs possessed essential structural elements required for Cas9 interaction including the repeat–anti-repeat region and three stem–loop structures. In vitro endonuclease assays demonstrated that the sgRNA targeting exon 22 of ERBB2 successfully cleaved its target DNA, producing fragments of 266 bp and 257 bp. Similar cleavage activity was observed in the two control sgRNAs, which generated fragments of 384 bp and 303 bp, and 486 bp and 201 bp, respectively. In contrast, the sgRNA targeting exon 25 of ERBB2 exhibited no detectable cleavage activity.CONCLUSION: The sgRNA targeting exon 22 of ERBB2 demonstrated effective DNA cleavage activity in vitro, whereas the sgRNA targeting exon 25 showed no endonuclease activity.KEYWORDS: CRISPR–Cas9, endonuclease, ERBB2, in vitro, breast cancer, RNP, sgRNA
Co-Authors Afifah, Salma Aulia Anindyajati ARTARINI, ANITA CATUR RIANI Christianto, Antonius DEBBIE SOEFIE RETNONINGRUM DESSY NATALIA Dirgahyu, Anugrah Prima ERNAWATI ARIFIN GIRI-RACHMAN Gunsi, Via Susana Meitha, Karlia Novia Syari Intan Putri Ayu Fajar RUKMAN HERTADI Wojciechowska, Gladys Emmanuella Putri