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Konstruksi Plasmid Rekombinan untuk Inisiasi Translasi Enhance Green Fluorescent Protein oleh Internal Ribosomal Entry Site HIV-1 Cintera Rahmagiarti; Silvia Tri Widyaningtyas; Budiman Bela
Media Penelitian dan Pengembangan Kesehatan Vol 28 No 2 (2018)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/mpk.v28i2.181

Abstract

Human immunodeficiency virus (HIV) is a virus that causes acquired immunodeficiency virus syndrome (AIDS). The HIV genome has a cap structure at 5’ and polyadenylation at 3’ on mRNA resulting in a translation initiation through scanning at 5'untranslated region (UTR). The Vpr protein produced during viral replication causes the 5'cap scanning to be inhibited so HIV-1 can directly recruit the ribosome at the start codon via internal ribosomal entry site (IRES). IRES activity is high at G2/M phase and highest expression in monocyte cell line (THP-1) and lymphocyte (HPB-ALL). The role of HIV IRES however, is not yet known in infection of nondividing cells by HIV-1. HIV-1 IRES and egfp from pcDNA5FRT/TO were amplified with PCR. The insert DNA (HIV-1 IRES_egfp) and pcDNA3.1(+) were digested with EcoRI and ApaI and then ligated. The verification was performed with PCR colonies, restriction verification, and sequencing. The size of insert DNA is 1067 bp while the vector is 5379 bp. E. coli transformed with DNA ligation produces 70 colonies, control of ligation produces 5 colonies, and negative control didn’t grow. 19 colonies contain recombinant DNA, restriction verification was of the appropriate size, and the sequence verification didn’t find any mutation. Therefore, the subcloning process pcDNA3.1_IRES HIV-1_egfp was successfully performed. Abstrak Human immunodeficiency virus (HIV) merupakan virus penyebab acquired immunodeficiency virus syndrome (AIDS). Genom HIV memiliki struktur cap di 5’ dan poliadenilasi di 3’ mRNA sehingga proses inisiasi translasi melalui pemindaian 5’cap pada struktur untranslated region (UTR) di 5’ mRNA HIV. Protein Vpr yang dihasilkan selama replikasi virus menyebabkan pemindaian melalui 5’cap terhambat sehingga HIV-1 dapat langsung merekrut ribosom pada kodon awal translasi melalui struktur internal ribosomal entry site (IRES). Aktivitas IRES tinggi pada fase G2/M dan ekspresi gen tinggi pada sel line monosit (THP-1) dan limfosit (HPB-ALL). Namun, peran IRES HIV-1 belum diketahui pada sel tidak membelah yang merupakan sel target pada infeksi HIV-1. DNA sekuen IRES HIV-1 dan egfp dari pcDNA5FRT/TO diamplifikasi dengan PCR. DNA sisipan (IRES HIV-1_egfp) dan pcDNA3.1(+) dipotong dengan EcoRI dan ApaI lalu DNA sisipan diligasi dengan pcDNA3.1(+). Verifikasi hasil klona dilakukan dengan PCR koloni, verifikasi restriksi, dan sekuensing. Restriksi DNA sisipan menghasilkan pita berukuran 1067 pb. Restriksi vektor plasmid menghasilkan pita berukuran 5379 pb. E.coli yang ditransformasi DNA ligasi menghasilkan 70 koloni, kontrol ligasi 5 koloni, dan kontrol negatif tidak tumbuh. 19 koloni terverifikasi mengandung DNA rekombinan, verifikasi restriksi memiliki ukuran sesuai, dan verifikasi sekuensing tidak terdapat perubahan basa. Oleh karena itu, proses subkloning pcDNA3.1_IRES HIV-1_egfp berhasil dilakukan.
Konstruksi Plasmid Pengekspresi Antigen Gag dan Protein Penghantar VP22 untuk Pengembangan Vaksin HIV-1 Melinda Remelia; Budiman Bela; Silvia Tri Widyaningtyas; Fera Ibrahim
Media Penelitian dan Pengembangan Kesehatan Vol 31 No 2 (2021)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/mpk.v31i2.3046

Abstract

The endogenous HIV-1 vaccine based on Gag protein is expected to stimulate the immune response of CD8+ T cells (cytotoxic). The Gag protein that has been produced by the E.coli prokaryote system is an exogenous antigen. The fusion of VP22 protein is expected to deliver Gag antigen into the cytoplasm of cell, observed by eGFP markers. Sequences of VP22 (114 pb), GagHIV-1 (1506 pb), and eGFP (733 pb) were inserted into the pQE80L, respectively. The recombinant protein was expressed in the E.coli system and purified by the Ni-NTA method. Antigen delivery fused with VP22 and eGFP was observed with fluorescence and confocal microscopy. The recombinant plasmid constructs of protein expression eGFP, VP22-eGFP, GagHIV-1-eGFP, VP22-GagHIV-1-eGFP were verified by DNA sequencing according to the reference. The recombinant plasmid constructs of Gag HIV-1-eGFP and VP22-GagHIV-1-eGFP still need to be optimized so they can be expressed in the E.coli system. The recombinant protein VP22-eGFP (27.02 kDa) was succesfully obtained and fluorescent green (entered) into the cytoplasm and nucleus of vero cells. In addition to the HIV-1 vaccine, this recombinant plasmids pQE80L-eGFP and pQE80L-VP22-eGFP also have the potential to be used as tools in the development of endogenous vaccines for another viruses/microbes. Abstrak Vaksin endogen HIV-1 berbasis protein Gag diharapkan dapat menstimulus respons imun sel T CD8+ (sitotoksik). Protein Gag yang telah diproduksi dengan sistem prokariota E.coli merupakan antigen yang bersifat eksogen. Fusi protein VP22 diharapkan mampumenghantarkan antigen Gag masuk ke sitoplasma sel, diamati dengan marker eGFP. Sekuens VP22 (114 pb), GagHIV1 (1506 pb), dan eGFP (733 pb) telah diinsersikan pada vektor pQE80L. Protein rekombinan diekspresikan pada sistem E.coli dan dipurifikasi dengan metode Ni-NTA. Penghantaran antigen yang difusikan dengan VP22 dan marker eGFP diamati dengan mikroskop fluoresens dan konfokal. Konstruksi plasmid rekombinan pengekspresi protein eGFP, VP22-eGFP, GagHIV1-eGFP, dan VP22-GagHIV1-eGFP telah diverifikasi dengan sekuensing DNA sesuai dengan sekuen referensi. Plasmid rekombinan pengekspresi GagHIV1-eGFP dan VP22-GagHIV1-eGFP masih perlu dioptimasi agar dapat diekspresikan di sistem E.coli. Protein rekombinan VP22-eGFP (27,02 kDa) telah berhasil diperoleh serta berpendar fluoresens hijau (masuk) ke sitoplasma dan nukleus sel vero. Selain vaksin HIV-1, plasmid rekombinan pQE80L-eGFP dan pQE80L-VP22-eGFP juga berpotensi dapat digunakan sebagai ‘tools’ dalam pengembangan vaksin endogen dari virus atau mikroba lainnya.
Construction of plasmids expressing recombinant B cell epitopes of PD1 Sofy Meilany; Andrijono Andrijono; Pauline Phoebe Halim; Budiman Bela
Health Science Journal of Indonesia Vol 10 No 1 (2019)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/hsji.v10i1.1848

Abstract

Latar Belakang: Pengobatan kanker di Indonesia umumnya menggunakan pengobatan dengan kemoterapi atau dengan operasi. Efek samping dari pengobatan ini antara lain adalah kerontokan rambut, mual dan penurunan berat badan. Saat ini sedang berkembang alternatif terapi kanker dengan menggunakan immunoterapi. Kemampuan sel kanker untuk menghindar dari sistem imun disebabkan adanya protein PD-1 pada sel T yang berikatan dengan ligannya PD-L1. Metode: Penelitian ini merupakan penelitian awal yaitu pembuatan rekombinan PQE PD-1 dan menggunakan bagian soluble dari PD-1 yang disebut dengan EP2PD1 yang akan digunakan untuk pembuatan antibodi monoklonal dan sistem pendeteksi antibodi monoklonal. Metode pembuatan rekombinan PD-1 dan EP2PD1 dengan cara penentuan sekuens epitop sel B yang paling imunogenik dilanjutkan dengan amplifikasi sekuen tersebut dengan PCR dan diligasi ke vektor pengekspresi PQE80. Hasil: Telah terbentuk konstruksi rekombinan PQE80 PD-1 dan PQEEP2PD1 yang diverifikasi menggunakan PCR koloni, pemotongan enzimatik dan sekuensing. Hasil penelitian menunjukkan bahwa epitop PD1 telah terklona ke PQE 80 dan tidak ditemukan mutasi dalam urutan asam amino. Kesimpulan: Konstruksi yang dibuat tidak mempunya mutasi dan dapat dilanjutkan untuk pembuatan antibodi monoklonal. Kata Kunci: PD1, Epitop, Kanker, Immunotherapy Abstract Background: Medications on cancer to date in Indonesia is mostly by surgical or chemotherapy, this type of medications is not always curing the patients. The side effect of the chemotherapy drugs sometimes more challenging such as hair loss, nausea and lost weight. One of the promising targets for cancer is using immune therapy. Cancer cells can avoid immune response by surprising immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. Immune check points like PD-1, PD-L1 are breakthrough therapies in oncology and this monoclonal antibody have been approved by the FDA for treatment. In this research we develop full PD-1 and part of PD1 sequence as an insert then we construct with plasmid PQE80L. This recombinant called PQE PD-1 and PQEEP2PD1. The aim of this study is to make recombinant which would be used to detect PD1 full clone monoclonal antibodies. Methods: In this study, we designed our recombinants using Indonesian HLA and others using in silico models, this prototype will not only cover Indonesian patients but also other country. Results: The result showed that the epitope sequence of PD1 has been clone to PQE 80 wt and verified using colony PCR, Enzyme Digestion and Sanger Sequencing. The Clone than will be expressed and injected to animal model to produce antibody. Conclusion: Construction of recombinant PQE PD-1 and PQE EP2PD1 are constructed without any mutation in the sequence, this recombinant can be used in the next study for protein expression of PQE PD-1 and PQE EP2PD1. Keywords: PD1, Epitope , Cancer, Immunotherapy
Cloning and expression of Human Papilloma virus type 16 L1 capsid protein in bacteria Silvia Tri Widyaningtyas; Sofy Meilany; Budiman Bela
Health Science Journal of Indonesia Vol 10 No 2 (2019)
Publisher : Sekretariat Badan Penelitian dan Pengembangan Kesehatan

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22435/hsji.v12i2.2442

Abstract

Latar belakang: Secara alamiah protein kapsid L1 Human Papillomavirus (HPV) tipe 16 dapat mengalami auto assembly untuk membentuk Viral like particle (VLP). Terkait dengan penelitian vaksin HPV, VLP dapat digunakan untuk berbagai keperluan seperti vaksin, pseudovirion atau SpyTag-Spycatcher. Penelitian ini ditujukan untuk mendapatkan plasmid rekombinan yang digunakan untuk produksi protein L1 HPV 16. Metode: Gen penyandi protein L1 HPV 16 diklona ke dalam vector pQE80L, suatu plasmid yang mengandung sistem ekspresi untuk prokariota. DNA penyandi HPV 16 L1 disisipkan pada situs restriksi BamHI dan Hind III plasmid pQE80L. Plasmid rekombinan yang mengandung gen L1 HPV 16dikonfirmasi menggunakan PCR dan analisis enzim restriksi. Lebih lanjut untuk memastikan bahwa gen rekombinan L1 HPV 16 dapat diekspresikan dalam prokariota, plasmid rekombinan ditransformasikan ke bakteri Escherichia coli BL21 (DE3). Bakteri diinduksi dengan Isopropyl β-D-1-thiogalactopyranoside (IPTG) dengan berbagai konsentrasi dan berbagai waktu inkubasi. Hasil: protein rekombinan L1, berat 56 kDa, telah berhasil diekspresikan dalam sistem prokariota. Protein rekombinan L1 dapat dimurnikan menggunakan TalonR dalam kondisi denaturasi. Kesimpulan: gen L1 HPV 16 telah dikloning ke dalam pQE80L dan berhasil diekspresikan dalam sistem prokariota. (Health Science Journal of Indonesia 2019;10(2):82-9) Kata kunci: L1, HPV 16, cervical cancer Abstract Background: Naturally Human Papillomavirus (HPV) type 16 L1 capsid protein can auto assemble to form Viral like particles (VLP). Concerning to vaccine development for HPV, VLP can be used for a variety of needs such as a vaccine, pseudovirion or SpyTag-Spycatcher. In this study, to obtain a vector expression that can be used in the production of HPV L1 protein, we cloned gene coding HPV 16 L1 protein into pQE80L a plasmid contains an expression system for prokaryote. Methods: The DNA coding HPV 16 L1 was inserted at BamHI and Hind III restriction sites of pQE80L plasmid. The recombinant plasmid containing the HPV L1 gene was confirmed using PCR colony and enzyme restriction. Further to ensure the recombinant HPV 16 L1 gene could be expressed in a prokaryote, the recombinant plasmid was transformed into bacteria Escherichia coli BL21 (DE3). The bacteria were induced with IPTG with various concentrations and various incubation time. Result: L1 recombinant protein, 56 kDa in weight, has successfully been expressed in prokaryote system. L1 recombinant protein can be purified using TalonR under denaturing conditions. Conclusion: L1 HPV 16 gene has been cloned into pQE80L and successfully expressed in prokaryote system. (Health Science Journal of Indonesia 2019;10(2):82-9) Keywords: L1, HPV 16, cervical cancer
Deteksi Acinetobacter baumannii Multiresisten Obat Penghasil Biofilm menggunakan Pewarnaan Berbasis Crystal Violet Ardiana Kusumaningrum; Iin Maemunah; Dimas Seto Prasetyo; Fera Ibrahim; Budiman Bela
The Indonesian Journal of Infectious Diseases Vol 5, No 2 (2019): The Indonesian Journal of Infectious Disease
Publisher : Rumah Sakit Penyakit Infeksi Prof Dr. Sulianti Saroso

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (120.703 KB) | DOI: 10.32667/ijid.v5i2.86

Abstract

Pendahuluan: Kasus infeksi terkait biofilm merupakan masalah besar pada dunia kesehatan karena sifat kekebalannya terhadap antibiotik dan respon imun, terutama pada kasus infeksi kronik akibat Acinetobacter baumannii. Saat ini terdapat berbagai metode deteksi pembentukan biofilm, namun pemeriksaan tersebut belum dilakukan secara rutin karena berbagai keterbatasan. Oleh karena itu, perlu dilakukan optimasi metode deteksi biofilm menggunakan bahan yang rutin tersedia di laboratorium serta mengukur proporsi sampel yang mengandung bakteri penghasil biofilm Metode: Desain penelitian ini adalah uji eksperimental. Pada penelitian ini dilakukan pengembangan deteksi pembentukan biofilm menggunakan metoda tabung microcentrifuse tube 1,5 ml berbahan polypropylene dengan zat warna berbasis crystal violet konsentrasi 0,1% dan 1%. Optimasi yang dilakukan meliputi media biakan, lama inkubasi, inokulum bakteri yang digunakan serta bahan pembilas. Isolat bakteri Pseudomonas aeruginosa ATCC 10145, A. baumannii ATCC 19606  dan Staphylococcus aureus ATCC 25923 digunakan sebagai kontrol positif dan kontrol negatif pemeriksaan yang dilakukan. Hasil: Pemeriksaan optimasi yang dilakukan, diperoleh kondisi optimal pembentukan biofilm menggunakan media Luria Bertani dengan besar inokulum 1 sengkelit penuh, lama inkubasi 30 jam dan pewarnaan dilakukan menggunakan crystal violet 0,1% serta bahan pembilas berupa PBS steril. Proporsi pembentukan biofilm pada A. baumannii multiresisten obat sebesar 55,3%. Kesimpulan: Metode deteksi pembentukan biofilm menggunakan metode tabung polypropylene yang dimodifikasi dan pewarnaan zat warna crystal violet 0,1% merupakan metode deteksi yang mudah dikerjakan, reproducible dan efisien, sehingga dapat dilakukan di laboratorium mikrobiologi klinik sederhana. Proporsi bakteri penghasil biofilm adalah lebih dari 50% A. baumannii resisten multiobat.
Prevalensi Antibodi IgG dan DNA Cytomegalovirus pada darah donor di unit transfusi darah Provinsi DKI Jakarta Ganjar Noviar; Ni Ken Ritchie; Budiman Bela; Yuyun SM Soedarmono
JHECDs: Journal of Health Epidemiology and Communicable Diseases Vol 3 No 1 (2017): JHECDs Vol. 3, No. 1, Juni 2017
Publisher : Balai Litbangkes Tanah Bumbu

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (531.23 KB) | DOI: 10.22435/jhecds.v3i1.1814

Abstract

Indonesia has not conduct regular screening test of CMV infection due to the lack of seropositive prevelance data information. However, seronegative CMV results is not an indicator of safe blood for transfusion, so that another test that serves as confirmation test for CMV DNA is required. The aim of this study is to obtain prevalence data of CMV IgG antibody positive, the prevalence of CMV DNA positive and to determine the effect of CMV IgG titers against CMV DNA in blood donors in UTD PMI DKI Jakarta. Cross-sectional method was used to test 113 blood donor samples which have met inclusion criteria. Screening for CMV IgG antibody was held using indirect method chemiluminescence immunoassay (ChLIA) by Liason® XL 10050 Chemiluminescence Analyzer and CMV DNA analysis using qPCR method for the detection of CMV UL 54 with a tool Roche Light Cycler 480 II. Results indicate positive prevalence of IgG CMV in 111 samples (98.23%), and negative CMV IgG in 2 samples (1.77%). Prevalence of CMV DNA positive donors is one sample (0.88%), 112 negative CMV DNA samples (99.12%) and Fisher's test results {P (0.982)> α (0.05)} showed no significant association between CMV IgG status with CMV DNA. CONCLUSIONS: UTD DKI Jakarta has a high prevalence of CMV IgG with low prevalence of CMV DNA.
Analisis Infektivitas Simian-Human Immunodeficiency Virus pada Peripheral Blood Mononuclear Cell Macaca fascicularis dan M. nemestrina Secara In Vitro Gede Eko Darmono; Silvia Triwidyaningtyas; Budiman Bela; Diah Iskandriati; Dondin Sajuthi; Joko Pamungkas
Jurnal Veteriner Vol 23 No 1 (2022)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (321.183 KB) | DOI: 10.19087/jveteriner.2022.23.1.16

Abstract

Development of human immunodeficiency virus type 1 (HIV-1) vaccines and anti-retroviral treatmentis currently hindered by the lack of models representing prominent symptoms of HIV-1 infections seen in humans. Simian-human immunodeficiency virus (SHIV) was constructed to resolve the limitations of SIVmac model and has been used in nonhuman primate model ofviral infections, particularly infections by the close relatives of HIV-1. Macaca fascicularis and M. nemestrina are being developed as model HIV/AIDS, by using chimeric virus SHIV produced by replacing the nucleotide structure of cyclophilin A binding region, vif gene and nef of HIV-1 with cyclophilin A binding region, vif gene and nef from SIV. The research aims to study the model of HIV/AIDS on nonhuman primates PBMC in vitro using SHIV. In particular, the study aims to obtain information about the capability of SHIV replication in PBMC of M. fascicularis and M. nemestrina. Results showed a cytopathic effect (CPE) in the form of multinucleated giant cells and expression of p24 protein in PHA-stimulated PBMC cultures of M. fascicularis and M. nemestrina after SHIV infection. The conclusion of this study is that SHIV can infect PBMC M. fascicularis and M. nemestrina in vitro based on CPE and expression of p24 protein.
Development of Recombinant Immunoblot Assay Diagnostic Test Based on HIV-1 in Indonesia Jeanne Elvia Christian; Silvia Tri Widyaningtyas; Budiman Bela
Molecular and Cellular Biomedical Sciences Vol 6, No 2 (2022)
Publisher : Cell and BioPharmaceutical Institute

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21705/mcbs.v6i2.251

Abstract

Background: High mutation rates in HIV-1 could affect the accuracy of diagnostic tests. Therefore, recombinant antigen that has an immunodominant and conserved region from HIV-1 need to be developed to detect HIV-1 infection in Indonesia.Materials and methods: The recombinant antigens comprise of Gag (p24), Pol and Env (gp41). Each antigens was expressed in the Escherichia coli expression system and purified using Ni-NTA chromatography. The reactivity of purified antigen against HIV antibodies was tested against a group of 50 HIV-positive plasma samples and 45 HIV-negative plasma samples in a recombinant immunoblot assay (RIBA) platform test. Moreover, 21 of 50 HIV-positive samples and 3 of 45 HIV-negative samples were also tested using HIV blot 2.2 to compare RIBA with a commercial western blot kit. Ten HBV-positive and 10 HCV-positive plasma samples were used to check cross-reactivity with HIV recombinant proteins in RIBA.Results: All HIV-positive samples (100%) tested with RIBA were reactive towards Gag (p24), Pol, Env (gp41). Otherwise, 3 of 21 HIV-positive samples assayed with HIV blot 2.2 were not reactive to Pol protein. All HIV-negative samples tested with RIBA and 3 HIV-negative samples tested with HIV blot 2.2 did not produce any bands of HIV antigens. Few HBV and HCV samples showed reactivity towards HIV recombinant proteins.Conclusion: Each recombinant protein, Gag (p24), Pol, Env (gp41), could be expressed and purified, as well as had reactivity to HIV-positive samples in RIBA test. Therefore, RIBA can be used as a diagnostic test to detect HIV-1 infection in Indonesia.Keywords: diagnostic, HIV-1, immunodominant, recombinant immunoblot assay (RIBA)
Computational Analysis of Chromophore Tripeptides FollowingFusion of Enhanced Green Fluorescent Protein and Cell-penetrating Peptides Widyaningtyas, Silvia Tri; Pratiwi, Ekawati Betty; Bela, Budiman
Makara Journal of Science Vol. 24, No. 4
Publisher : UI Scholars Hub

Show Abstract | Download Original | Original Source | Check in Google Scholar

Abstract

Cell-penetrating peptides (CPPs) are small peptides that can transfer other materials into a cellular compartment. In this research, we studied the effect of fusion of new CPPs to the N-terminal of enhanced Green Fluorescent Protein eGFP on the ability of the latter to fluoresce. Results showed that the recombinant protein CPPs-eGFP could be successfully expressed in Escherichia coli. In contrast to E. coli expressing wild-type eGFP, which could fluoresce under ultraviolet (UV) or visible light, E. coli expressing CPPs-eGFP lost their ability to fluoresce. PyMol, a molecular visualization system, revealed that fusion of the new CPPs to the N-terminal of eGFP alters interactions between chromophore-forming tripeptides and the adjacent amino acids of other tripeptides. Disrupting peptide interactions induced structural changes in eGFP that caused it to lose its fluorescence ability. We suggest performing computational analyses to predict the biological function of new fusion proteins prior to starting laboratory work.
Expression and the Functional Study of Fusion Proteins α-Amylase and Hemolysin- αas an Application in Biofilm Polysaccharide Degradation Sugiarta, Gede Yuda; Wiseso, Anggoro; Sari, Siska Yuliana; Kamila, Etri Dian; Geraldine, Vanessa; Christina, Diana; Hanifi, Muhammad; Satyapertiwi, Dwiantari; Hertanto, Robby; Bela, Budiman; Yohda, Masafumi; Sahlan, Muhamad
Makara Journal of Technology Vol. 20, No. 3
Publisher : UI Scholars Hub

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Abstract

Biofilm is an aggregate of consortium bacteria that adhere to each other on a surface. It is usually protected by the exopolysaccharide layer. Various invasive medical procedures, such as catheterization, endotracheal tube installation, and contact lens utilization, are vulnerable to biofilm infection. The National Institute of Health (NIH) estimates 65% of all microbial infections are caused by biofilm. Periplasmic α-amylase (MalS) is an enzyme that hydrolyzes α-1, 4- glicosidic bond in glycogen, starch, and others related polysaccharides in periplasmic space. Another protein called hemolysin-α (HlyA) is a secretion signal protein on C terminal of particular peptide in gram negative bacteria. We proposed a novel recombinant plasmid expressing α-amylase and hemolysin-α fusion in pSB1C3 which is cloned into E.coli to enable α-amylase excretion to extracellular for degrading biofilm polysaccharides content, as in starch agar. Microtiter assay was performed to analyze the reduction percentage of biofilm by adding recombinant E.coli into media. This system is more effective in degrading biofilm from gram positive bacteria i.e.: Bacillus substilis (30.21%) and Staphylococcus aureus (24.20%), and less effective degrading biofilm of gram negative i.e.: Vibrio cholera (5.30%), Pseudomonas aeruginosa (8.50%), Klebsiella pneumonia (6.75%) and E. coli (-0.6%). Gram positive bacteria have a thick layer of peptidoglycan, causing the enzyme to work more effectively in degrading polysaccharides.
Co-Authors Ade P.R. Simaremare Andi Yasmon Andrijono Andrijono Anggraini Alam Anis Anis Ardiana Kusumaningrum Armi Susandi Aroem Naroeni Aroem Naroeni Ascobat, Purwantyastuti ASRI SULFIANTI Beti Ernawati Dewi Cahyani, Hesti Pramesti Christina, Diana Cintera Rahmagiarti Diah Iskandriati Dian Amirulloh Dimas Seto Prasetyo Dondin Sajuthi Elisna Syahruddin Evy Yunihastuti FERA IBIRAHIM Fera Ibrahim Firdaus Sirait Ganjar Noviar Gede Eko Darmono Geraldine, Vanessa Hanifi, Muhammad Hartiyowidi Yuliawuri Hartono, Soni Herna Herna Herna, Herna Hertanto, Robby Ibnu Agus Ariyanto Iin Maemunah Ingrid S. Surono Jeanne Adiwinata Pawitan Jeanne Elvia Christian Kamila, Etri Dian Ketut Tuti Parwati Lili Indrawati Maria Lina R. Maria Lina Rosilawati Melinda Remelia Muhayya, Syarifah Raisha Murdani Abdullah Mursinah Mursinah Mursinah Mursinah Ni Ken Ritchie Ni Nengah Dwi Fatmawati Nia Kurniati Nuzli Fahdia Mazfufah Pamungkas, Joko Pauline Phoebe Halim Pramita Gayatri Pratiwi, Ekawati Betty Radiana Dhewayani Antarianto SAHLAN, MUHAMAD Sari, Siska Yuliana Satyapertiwi, Dwiantari Silvia Tri Widyaningtyas Silvia Tri Widyaningtyas Silvia Tri Widyaningtyas Silvia Tri Widyaningtyas Silvia Tri Widyaningtyas Silvia Triwidyaningtyas Sofy Meilany Subangkit Subangkit Subiantistha, Tanaya Sugiarta, Gede Yuda SUWIJIYO PRAMONO Utami, Mardhah Sastri VIVI SETIAWATY Widianingtyas, Silvia Widyaningtyas, Silvia Tri Wiseso, Anggoro Yohda, Masafumi YULIANTY MUHAYAR Yuliar Budi Hartanto Yusmaniar Yusmaniar Yuyun SM Soedarmono Yuyun Soedarmono Zakiudin Munasir