LAPORAN PROGRAM PENINGKATAN PUBLIKASI … · LAPORAN PROGRAM PENINGKATAN PUBLIKASI INTERNASIONAL MAHASISWA S3 ... “Karakterisasi dan identifikasi bakteri yang berasosiasi dengan

LAPORAN

PROGRAM PENINGKATAN PUBLIKASI

INTERNASIONAL MAHASISWA S3

TAHUN ANGGARAN 2013

Oleh

RINA ANITA MOGEA PASCASARJANA UNIVERSITAS BRAWIJAYA

KEMENTRIAN PENDIDIKAN DAN KEBUDAYAAN DIREKTORAT JENDERAL PENDIDIKAN TINGGI

DIREKTORAT PENDIDIKAN DAN TENAGA KEPENDIDIKAN 2013

LEMBAR PENGESAHAN LAPORAN PROGRAM SANDWICH

OLEH

NAMA MAHASISWA : RINA ANITA MOGEA NIM : 117090100111015 PROGRAM STUDI : BIOLOGI PT ASAL (TEMPAT BERTUGAS)

: UNIVERSITAS NEGERI PAPUA

PT TUJUAN SANDWICH : RITSUMEIKAN UNIVERSITY NEGARA TUJUAN : JAPAN

PROMOTOR PESERTA SANDWICH

. ( Dr. SUHARJONO, MS) ( RINA ANITA MOGEA )

MENGETAHUI, DIREKTUR PROGRAM PASCA UB

Prof. Dr. Ir. SOEMARNO, MS. NIP. 19550817 198003 1 003

PROGRAM PASCASARJANA UNIVERSITAS BRAWIJAYA

MALANG 2013

Kata Pengantar

Segala Puji syukur kepada Tuhan Yang Maha Esa atas Kasih dan RahmatNya, kegiatan sandwich-like program 2013 telah kami ikuti dengan baik. Kegiatan ini merupakan salah satu penunjang penyusunan disertasi ini telah terlaksana selama tiga bulan di Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University, Japan. Secara garis besar kegiatan-kegiatan yang terutama berupa karakterisasi sampel penelitian dan penyusunan publikasi ilmiah berlangsung dengan baik sesuai yang direncanakan berkat dukungan berbagai pihak.Untuk itu pada kesempatan ini kami mengucapkan terima kasih kepada:

1. Kementrian Pendidikan Dan Kebudayaan RI atas kesempatan dan dukungan dana yang diberikan

2. Prof. Ir. Sukoso, M.Sc, P.hD atas dukungan, arahan, bimbingan dan rekomendasi yang diberikan

3. Dr. Luchman Hakim, Ketua Program Doktor Biologi FMIPA Universitas Brawijaya, atas dukungan dan rekomendasi yang diberikan

4. Dr Suharjono, MS sebagai Pembimbing dukungan, arahan, bimbingan dan rekomendasi yang diberikan

5. Direktur pascasarjana UB beserta staf atas dukungan administratif dan talangan dana yang diberikan

6. Prof. Motoki Kubo atas dukungan dan bimbingannya dalam pelaksanaan Sandwich- Like Program .

7. All members of Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University atas kerjasamanya selama sandwich berlangsung.

Malang, 22 Mei 2014

Peserta

Rina Anita Mogea.

Bab 1. Pendahuluan

a. Latar belakang

Pemerintah Indonesia melakukan berbagai cara untuk mempersiapkan

sumberdaya manusia Indonesia yang berkualitas dan mempunyai daya saing

internasional melalui perguruan tinggi, untuk mencapai hal tersebut perlu juga untuk

meningkatkan kualitas dosen di perguruan tinggi. Para dosen di PT kebanyakan

melanjutkan studi pascasarjana di dalam negeri. Untuk menambah wawasan para

dosen yang studi S3 diberikan kesempatan untuk mengikuti program Sandwich-S3

luar negeri. Saya sebagai mahasiswa program pascasarjana di Universitas Brawijaya

memanfaatkan kesempatan yang ditawarkan oleh Dikti, dengan semangat serta

dorongan dan dukungan dari pembimbing, mengajukan usulan untuk mengikuti

program tersebut.

Penyusunan disertasi untuk memenuhi persyaratan kelulusan dalam pendidikan

S3 perlu dilakukan penelitian. Adapun judul penelitian yang sedang dilakukan

“Karakterisasi dan identifikasi bakteri yang berasosiasi dengan Ascidian” dalam

melakukan penelitian ini kami mengalami kendala yaitu keterbatasan alat

karakterisasi berdasarkan karateristik filogenik untuk menentukan suatu spesies

mikroba dan identifikasi ini berdasarkan informasi genomic yaitu membandingkan

sekuen-sekuen DNA atau RNA. Salah satu jalan keluar yang ditawarkan oleh

Direktorat Jenderal Pendidikan Tinggi, Direktorat Pendidikan dan Tenaga

Kependidikan yaitu memberi kesempatan bagi dosen yang sedang menempu

pendidikan S3 di dalam negeri untuk melakukan magang di perguruan tinggi atau

institusi riset luar negeri selama minimum empat bulan melalui Program Sandwich-

S3 luar negeri. Beragam kegiatan yang mendukung disertasi dapat dilakukan dalam

program tersebut dengan dukungan dana yang berupa : biaya hidup, biaya buku, dan

asuransi.

b. Tujuan Kegiatan 1. Memperoleh hasil riset dengan jalan melakukan karakterisasi dengan

menggunakan teknik berbasis PCR, instrument Kodak ID, 3.5 imaging

software, automatic DNA sequencer.

2. Menjalin komunikasi dan kerjasama di bidang ilmiah di tingkat internasional

3. Berkonsentrasi menyusun publikasi

c. Manfaat Kegiatan

1. Pelaksanaan penelitian lebih lancar

2. Dapat melakukan kolaborasi di bidang penelitian

3. Dapat mengetahui teknik penulisan artikel untuk jurnal internasional.

Bab II. Rencana Kegiatan

a. Tempat Kegiatan Di luar Negeri Kegiatan sandwich dilakukan di Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University, Japan. Selama mengikuti kegiatan sandwich di Ritsumeikan University, kami dibimbing oleh Prof . Motoki Kubo, PhD.

b. Waktu Kegiatan Kegiatan dilakukan selama 3 bulan, mulai 18 Oktober hingga 30 Desember 2013.

c. Lingkup kegiatan 1. Melakukan karakterisasi sampel meliputi ekstrak genom dna bakteri,

menghitung DNA bakteri dan jumlah sel bakteri dalam kultur, PCR dan sekeunsing.

2. Penulisan draf paper untuk jurnal dengan topic “Phylogenetic analysis of bacteria with antimicrobial activities from the Doreri of gulf Manokwari associated with ascidian Phallusia julinea “

3. Mengikuti seminar international di Kumamoto Universty sebagai pembawa materi

d. Data singkat pendamping di luar Data pendamping di luar negeri Nama : Prof Motoki Kubo Degree : Doctor of Engineering, University of Osaka (1992) Education Background

• B.S in Engineering, Hiroshima University (1983) • M.S in Engineering, Hiroshima University (1985)

• Dr in Engineering, Osaka University (1992) Career History 1994 – Ilinois State University School of Medicine Ministry of Education overseas

researcher 04/1997 - 03/2002. Associate Professor, Faculty of Science and Technology

Ritsumeikan University.

04/2002 - 03/2008. Science, Ritsumeikan University. Professor 04/2008 – now. Ritsumeikan University Life Science. Professor. Teaching Achievement

• Survey Report on biological engineering education in University. 1996. Journal of Biotechnology, Japan.

• Biological engineering education and substantiation of graduate education. Journal Biotechnology, Japan, Vol 88 No 11, Page 583-584,2010.

Paper • Distribution of Hydrocarbon Degrading Bacteria in the Soil Environment and

their Contribution to Bioremediation. 2013. Applied Biochemistry and Biotechnology. Vol 170 page 329-339.

• Soybean as a nitrogen sipplier. 2013. In Teck, Edited by James E. Board. Page 49-60.

• Further stabilization of Leu155 mutant thermolysins by mutation of an autodegradation site. 2012. Applied Biochemistry and Biotechnology. 166, 735-743.

• Analysis of peptide uptake and location of root hair-promoting peptide accumulation in plant roots. 2011. Peptide Science.

• Isolation and characterization of an enther-type polyurethane-degrading microorganism,and analysis of degradation mechanism by Alternaria sp. 2010. J. Appl. Microbiol. 108, 1946-1953.

• Utilization of biomass based on biorefinery-Development of novel bioactive peptides from soybean waste. 2009. Research Trends. 4, 83-91.

• Phylogenetic analysis of long-chain hydrocarbon-degrading bacteria and evaluation of their hydrocarbon-degradation by the 2,6-DCPIP assay. 2008. Biodegradation. 19, 749-757.

• Construction of an efficient bioremediation system for petroleum hydrocarbon-contaminated soils using specific hydrocarbon-degrading bacteria and bacterial biomass monitoring. 2008. Soil Contamination Research Trends,Editor J. B Dominguez, Nova Science Publishers Inc. New York.

• Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment. 2007. J.Biotechnol.Bioeng.

• Culture condition of Pseudomonas aeruginosa F722 for biosurfactant production. 2006. Biotechnology and Bioprocess Engineering. 11, 471-476.

• Degradation pathways of cyclic alkanes in Rhodococcus sp.NDKK48. 2005. Appl. Micobiol. Biotechnol. 66, 92-99.

• Microbial hydroxylation of indole to 7-hydroxyindole by Acinetobacter calcoaceticus strain 4-1-5. 2004. Biosci.Biotech.Biochem. 68, 1167-1169.

• Biodegradation enhancement of the mixture of kerosene and diesel by using biosurfactant from Pseudomonas aeruginosa F722. 2003. J.Biotechnol.Bioeng. 18, 529-535.

• Biodegradation of crude oil and petroleum products by crude oil-degrading microorganisms. 2002. Kor. J. Biotechnol. Bioeng. 17, 247-254.

• Cloning and phylogenetic analysis of two different bphC genes and bphD gene from a PCBs-degrading bacterium, Psudomonas sp. strain SY5. 2001. J. Microbiol. Biotechnol. 11, 668-676.

• Analysis of biosurfactant from Bacillus sp. ODTY34. 2000. Memories of the Institute of Science & Engineering, Ritsumeikan Univ. 59, 99-111.

• Mechanism of thermostability in thermolysin - analysis of subsite S2 mutant enzymes of thermolysin. 1991. Lett. in Appl. Microbiol. 28, 431-434.

• Isolation and characterization of polychlorinated biphenyls (PCBs) degrading bacteria from a municipal sewage treatment plant. 1998. Environ. Eng.Res. 3, 67-78.

• The present study of advanced engineering course in chemical field of Japanese national college technology. 1997. Numazu Coll. Technol. Res. Ann. 31, 63-71.

• A novel use of biomass resources. 1996. Bio Industry. 13, 44-51. • Alteration of specific activity and stability of thermostable neutral protease by

site-directed mutagenesis. 1992. Appl. Environ. Microbiol. 58, 3779-3783.

• Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus. 1988. J. Gen. Microbilol. 134, 1883-1892.

Academic society membership

• Environmental biotechnology society

• Japanese biochemical society

• Japanese society od soil science and plant nutrition • American society of microbiology

• Japan society for bioscience, biotechnology and agrochemistry • Society for biotechnology, Japan.

Subjech of research

• Studies on the (fat and high salt) liquid waste disposal • Studing on the new use of biomass resources

• Study on oil degradation and protease from microorganism.

Awards

• Watanabe Mitsuhiko Invention Award 03/2010 • Sapporo Bioscience Foundation Award a research grant in analysis of plant

growth promotin peptides from biomass resources. 2009.

e. Deskripsi rincian rencana kegiatan.

Judul : Aktifitas antimikroba dari bakteri yang berasosiasi dengan ascidian.

Rencana Kegiatan

Bakteri laut merupakan sumber senyawa bioaktif baru yang banyak menjadi perhatian sekarang ini. Bakteri laut yang miskin nutrisi banyak dijumpai hidup dengan cara berasosiasi dengan organisme laut bentik, seperti ascidian. Asosiasi bakteri dengan organisme laut dapat mempengaruhi proses metabolisme bakteri, hal ini terjadi karena kompetisi hidup yang tinggi antara bakteri dan keterbatasan nutrisi (Mearns-Spragg et al., 1998). Bakteri dapat menghasilkan senyawa bioaktif untuk berbagai aktivitas biologis, misalnya antimikroba. Produksi senyawa antimikroba merupakan salah satu cara bakteri beradaptasi terhadap lingkungan dalam upaya bakteri mempertahankan diri dari bakteri lain dan inangnya (Penesyan et al., 2010). Data di atas menunjukkan bahwa ada simbion yang teridentifikasi positif menghasilkan komponen bioaktif yang sama dengan inangnya. Dengan demikian penelitian ini dilakukan untuk mencari bakteri indigenous yang bersimbion dengan ascidian dari papua dan berpotensi sebagai penghasil antimikroba terhadap mikroba patogen.

Skema kegiatan

No

Rencana kerja

Kegiatan

Waktu

Lokasi kerja

1 Langka 1: yang telah dikerjakan yaitu memperoleh isolat bakteri indegenus yang berasosiasi dengan

Mengisolasi bakteri yang dapat menghambat mikroba patogen

Laboratorium mikrobiologi FMIPA UB

ascidian

Mengumpulkan bakteri yang memiliki aktivitas antimikroba

November 2012 – Juli 2013

2 Langka 2 yang akan dilakukan di laboratorium bioengineering, Fakultas MIPA Universitas Ritsumeikan yaitu identifikasi bakteri

Identifikasi bakteri menggunakan 16S rDNA

Oktober – Desember 2013

Ritsumeikan

University

f. Deskripsi Luaran (output) kegiatan :

1. Data hasil genom DNA bakteri,

2. Data hasil kuantitas DNA dan jumlah sel bakteri dalam kultur

3. Data PCR dan analisa sekuens bakteri.

g. Deskripsi Dampak (outcome) kegiatan :

1. Memperdalam kajian dalam penulisan disertasi

2. Menambah wawasan ketrampilan dalam pengerjaan DNA bakteri

3. Dapat bertukar pikiran dan pengalaman dengan peneliti dari berbagai Negara.

Bab III. Pelaksanaan Kegiatan

a. Tahap Persiapan Kegiatan. Adapun tahap persiapan kegiatan meliputi tahapan sebagai berikut pada tabel dibawah ini. No Tanggal/Bulan Kegiatan 1 Desember 2012 Mengikuti sosiaslisasi sandwich dari dikti 2 Februari 2013 1. Mempersiapkan research plan yang akan

dilakukan di universitas tujuan 2. Melakukan komunikasi via email dengan mentor

di luar negeri untuk mendapatkan LoA 3 Maret 2013 1. Mengisi form A

2. Melengkapi dokumen-dokumen yang dibutuhkan 3. Menyerahkan dokumen dan kelengkapannya ke

secretariat pasca UB 4 17 April 2013 Mengikuti wawancara seleksi sandwich dari dikti 5 13 Mei 2013 Memperoleh LoA dengan pembimbing dari

Ritsumeikan University 6 18 Mei 2013 Pengumuman hasil seleksi wawancara program

sandwich-like tahun 2013 sesuai surat dari DIKTENDIK Dikti No. 699/E4.4/2013, tanggal 15 Mei 2013. • Melengkapi persyaratan keberangkatan ke luar

negeri yang meliputi: 1. LoA terbaru 2. Daftar riwayat hidup 4 rangkap 3. Pasfoto berwarna ukururan 4x6 (8 lembar) 4. Permohonan paspor dinas (6 rangkap) 5. Rencana keberangkatan (tanggal

keberangkatan, mulai s .d selesai) 6. Rencana biaya hidup 7. Fotokopi karpeg 8. Mengirimkan persyaratan tersebut ke

Kasubdit Kualifikasi Ditjen Pendidikan Tinggi Gedung D, Lt V, Jl. Jendral Sudirman, Pintu Satu-Senayan. Jakarta.

7. 29 Juni 2013 Orientasi pembekalan keberangkatan penerima beasiswa Peningkatan Publikasi Internasional (sandwich-like) luar negeri Ditjen Pendidikan Tinggi tahun 2013.

8 Juli 2013 Mempersiapkan surat-surat yang diperlukan oleh

Perguruan Tinggi Tujuan di luar negeri yaitu recommendation letter, declaration of understanding, research plan, application form for research student, bkc international house room request, dan coe application form.

9 Agustus 2013 Mendapatkan surat PKPI Scholarship dari dikti 10 September 2013 Mengirimkan kelengkapan surat ke perguruan tinggi

tujuan yaitu Ritsumeikan University. 11 Oktober 2013 1. Menerima CoE

2. Mengurus visa terbit tanggal 11 Oktober 2013 3. Melakukan reservasi tiket dengan galaxy holiday

tour & travel 4. Memperoleh tiket tanggal 17 Oktober 2013 dan

berangkat menuju Osaka-Japan b. Tahap Pelaksanaan Kegiatan

Tahap pelaksanaan kegiatan Program Peningkatan Publikasi Internasional (Sandwich-Like) tahun 2013 di Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University, Japan. Kegiatan ini berlangsung selama tiga bulan dari 20 Oktober s/d 30 Desember 2013).

c. Tahap Akhir Kegiatan

4. Akhir dari kegiatan ini yaitu mempresentasikan hasil yang dikerja selama magang di laboratorium bioengineering di working group Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University, pembuatan laporan kegiatan sandwich-like 2013 dan penyiapan materi seminar sandwich-like 2013 serta penyiapan draf jurnal internasional dengan judul “Phylogenetic analysis of bacteria with antimicrobial activities from the Doreri of gulf Manokwari associated with ascidian Phallusia julinea “ .

Bab IV. Penutup

a. Kesimpulan

Adapun yang dapat disimpulkan dari kegiatan sandwich-like yang telah dilakukan selama 3 bulan, bahwa kegiatan ini telah berlangsung sesuai dengan rencana. Peserta telah melakukan kegiatan karakterisasi dan identifikasi bakteri yang berasosiasi dengan ascidian dengan peralatan dan bahan media yang tersedia di Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University dan menyusun draft publikasi yang selanjutnya akan didiskusikan dengan promotor di Indonsia untuk di submit di salah satu jurnal internasional. Peserta juga memperoleh pengalaman yang bermanfaat dalam ketrampilan laboratorium.

b. Saran Program ini kiranya dapat berlanjut terus karena sangat menolong mahasiswa S3 dalam menyelesaikan studinya. Hal semacam ini dapat meningkatkan kompentensi mahasiswa memperoleh akses informasi, sarana dan prasarana yang lebih lengkap. Tetapi kiranya sistem administrasi pengelola program ini dan perguruan tinggi asal peserta hendaknya ditingkatkan supaya keterlambatan surat-surat yang dibutuhkan oleh perguruan tinggi tujuan bisa dihindari karena masalah ini mengganggu kelancaran pelaksaan program.

Daftar Pustaka

Mearns-Spragg, A., Bregu, M., Boyd, K.G., Burgess, J.G. 1998. Cross-species Induction and Enhancement of Antimicrobial Activity Produced by Epibiotic Bacteria from Marine Algae and Invertebrates, after Exposure to Terrestrial Bacteria. Letter Applied Microbiology 27:142-146.

Penesyan, A., Kjelleberg, S and Egan, S. 2010. Development of Novel Drugs from Marine Surface Associated Microorganisme. Marine Drugs 8:438-459.

L A M P I R A N

1. Surat-surat Ijin yang mendukung keberangkatan melaksanakan kegiatan a. Surat rekomendasi pembimbing dalam negeri b. Surat Ijin dari Direktur Pascasarjana UB

特 別 研 究 学 生 推 薦 書

Recommendation Letter for Research Student

※在学大学院の指導教員が記入してください。

Please have this form completed by your supervisor.

１．立命館大学特別研究学生としての推薦事由及び在学中の研究状況

Please indicate the reason for recommending that Rina Anita Mogea

(name) be accepted as a Research Student at Ritsumeikan Univer

sity and provide a description of the research conducted by the applicant at their hom

e institute.

I am glad that she follow this program cause with discussion and laboratory activ

ities on Ph.D research problems in the Ritsumeikan University is surely a good o

pportunity to improve her capability both as a lecture and researcher, as well a

s helping her completing the Ph.D dissertation. Until this day

she has done to isolation and chosen of bacteria can inhibit microbial pathoge

n.

She told me that her want to identification bacteria based on 16S rDNA after th

at she analysis

compound bioactive from bacteria.

２．本人の健康状況等その他特記事項

Other comments regarding the applicant’s health condition, etc.

I think her health condition is good to doing research in japan

日付: 2013 年 7 月 14 日

Date: (Year) (Month) (Day)

2. Fotocopy pasport a. halaman identitas pada paspor b. halaman keberangkatan c. halaman kepulangan

3. Dokumen Output Kegiatan a. Draf manuskrip paper untuk publikasi b. Resume kegiatan analisis laboratorium dan data c. Rincian kegiatan selama sandwich

Phylogenetic analysis of bacteria with antimicrobial activities from the Doreri of gulf Manokwari associated with ascidian Phallusia julinea.

Rina A. Mogea 1), Suharjono 2), Tri Ardiyati 2), Sutiman Samino 2) Motoki Kubo3)

1) PhD student, Department Biology Faculty Science Brawijaya University-Indonesia 2) Department Biology Faculty Science Brawijaya University-Indonesia

3) Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University

Abstract

Objective : to observed isolation of bacterial from ascidian, screening the isolated for their antimicrobial activity, identification of bacteria for biochemical and molecular methods like 16S rDNA sequencing. Methods: The isolate and screening were carried out by different methods such as the method of double layer, paper disc diffusion method and screening of supernatant for antimicrobial substance production. Result : A Pseudomonas aeruginosa strain ZC6 was isolated from ascidian Phallusia julinea and the bacteria was identified by 16S rDNA analysis. The sequencen of the bacteria was submitted in DDBJ with accession number (KJ184550). This bacteria can produce bioactive compound like antimicrobial. Conclution: The result from this study indicate the potential of bacterial to produce antimicrobial compound and can be using for many applications and must be better exploration.

Keywords: ascidian Phallusia julineaI, antimicrobial activity, Pseudomonas aeruginosa, 16S rDNA, supernatan.

1. Introduction

Marine organisms have long known as a rich source for natural products. Produce a variety of marine natural products consisting of primary and secondary metabolites. The number of natural products isolated from marine organisms increased rapidly, and now more than 18,000 compounds, and each year found hundreds of new compounds. Most of these bioactive compounds have been extracted from sponges, ascidians, bryozoans, algae, anemones, molluscs and other organisms. These compounds can be developed as antimicrobial drugs, anticancer,

antiviral and anti-HIV (8). The marine organisms have specific relationship with numerous associated microorganisms and these are very important for ecology (1).

One of Indonesia marine resource potential as providers of raw materials for the pharmaceutical industry is ascidians or tunicates. Ascidian is a rich biota of bioactive compounds that can be used as a drug or medicinal substance. Some bioactive components that exist in ascidians include antitumor, antiviral, immunosuppressants, and cytotoxic (2). Ascidians are marine filter feeders

with a water-filled, sac-like body structure and two tubular opening known as siphons. Microorganisms have symbiotic relationship with ascidians cause their nutrient-rich and host-associated environments forming unique niche for microorganism exploitation.

Marine bacteria are a source of new bioactive compounds is much concern today, many studies have shown that bacteria have the ability to produce its. Bacteria can produce bioactive compounds for a variety of biological activities, such as antimicrobial (4,5,6,7,9,11). Production of antimicrobial compounds is one way bacteria adapt to the environment cause competition among microbes for space and nutrients in marine environment is a powerful selection pressure that give away marine microorganism to produce bioactive compound (12). Based on the above research was conducted isolation of marine bacteria contained in Ascidian Phallusia julinea. Parameters observed for this study was isolation of bacterial from ascidian, screening the isolated for their antimicrobial activity, identification of bacteria for biochemical and molecular methods like 16S rDNA sequencing.

2. Materials and methods 2.1. Isolation bacteria

Ascidian P julinea sample was collected from the Doreri of gulf Manokwari. Research station is an area of coral reef slope at a depth of 3-15 meters. The size of P julinea is seven centimeters in length were taken at a depth of 10 m at high tide in the

waters of Manokwari. Surface samples were rinsed with sterile seawater. For the isolation of bacteria taken from ascidian the first taken from the entry siphons, siphons out and the center tunic by using a sterile swab and then homogenized in 10 ml PBS. Suspension homogenization result suspension diluted serially in PBS. Suspension each dilution rate of 75 µl poured and leveled in the medium Sea Water Complete (SWC) with the composition of the medium 5 g / l bacto peptone, 1 g / l extrat yeast and 3 ml / l glycerol, 15 g/l bacto agar and incubated at 300C for 24 hours. Each colony isolates purified by repeatedly scraping the quadrant in SWC medium to obtain single colonies were pure and subcultured on SWC slants. The bacterial isolates so obtained were stored at 40C, for further study. The isolated strain was identified morphologically (shape, gram staining, spore staining, pigmentation, margin, elevation and motility).

2.2. Screening of isolate bacteria for antimicrobial activity

In primary screening, determination of the antimicrobial activity of pure isolates was done by the method of double layer (3) using four microbial test straind. They were methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, enteropathogenic Escherichia coli K-11 (EPEC K-11) and Candida albicans (Clinical isolate). The step for method double layer after obtained pure isolated first step pour SWC agar on plate until harden and than pour SWC agar contain clinical isolate. The

bacterial isolate from the SWC slants were spotted on SWC agar plates seeded with actively growing cells of clinical isolate. The plates were incubated for 24 hours at 300C. The resulting bioactive antimicrobial compounds indicated by the formation of clear zones around colonies of pure isolates. For microbial testing of Candida albicans media used were Potato Dextrose Agar (PDA) and another using SWC medium. After get the isolate and identified with biochemically using Microbact Kit 12A and 12B.

2.3. Screening of supernatant for antimicrobial compound production Supernatant from marine bacteria contained in Ascidian P julinea having more antimicrobial activity than the other isolates were tested for its antimicrobial substance production. The overnight grown culture of marine bacteria grown in SWC broth were centrifuged at 10000 rpm for 10 minutes and supernatant was collected. The overnight cultures of the clinical isolate in Nutrien broth and Potato Dextrose broth were uniformly swabbed on the sterile SWC agar plate and PDA plate using sterile cotton swabs. Take the paper disc with size 6mm by sterile tweezers into the 50 µl of the supernatant. Put the paper disc on SWC agar plate and PDA plate. The plates were incubated without

inverting for 24 hours at 300C and the zone of inhibition was noted.

2.4. Identification of bacterial strains

The bacteria with wide antimicrobial spectrum were identified to the species level by PCR amplification of the 16S-rDNA gene, BLAST analysis and comparison with sequences in the GenBank nucleotide database. Specifically, the 16S-rDNA using universal forward primer fD1 (5’AGAGTTTGATCCTGGCTCAG-3’) and reverse primer 1 rP2 (5’ – ACGGCTACCTTGTTACGACTT-3’). The PCR products were purified by QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Sequence data were analyzed by comparison with 16S rRNA genes in the GenBank database. The nearest relative of the organism was obtained by BLAST searches and aligned with close relatives using the Clustal W software. A phylogenetic tree was constructed with MEGA version 5.0 using maximum likehood.

3. Result

3.1. Isolation bacteria Marine bacteria were isolated

from ascidian P julinea from the Doreri of gulf Manokwari using SWC agar by serial dilution spread plate method. After incubation for 24 hours and get isolate colonies. Five bacteria were isolated based on their morphological (Table.1)

Table 1. Morphological of isolate bacteria from marine Isolate bacteria

Shape elevation margin pigmentation Gram staing

Spore staing

Motility

Pj1 Round Raised smooth green negative - Motile Pj2 Round Flat smooth orange negative - Non

motile Pj3 Round convex smooth cream positive + Non

motile Pj4 Round convex smooth cream negative - Non

motile Pj5 Round raised smooth green negative - Non

motile

3.2 Screening of isolate bacteria for antimicrobial activity

All of the isolates bacteria were test for their ability to produce

antimicrobial compound against four clinical isolate used double layer method. Out of the five isolate, only one isolate bacteria showed inhibitory activity against clinical isolate of microbial pathogens after 24 hours of incubation. One bacteria was isolated based on its identified with biochemically using Microbact Kit 12A and 12B.

Table 2. Identification of isolate Pj1

No Biochemical tests Results 1 Oxidase + 2 Motility + 3 Nitrate + 4 Lysine - 5 Ornithine - 6 H2S - 7 Glucose - 8 Mannito + 9 Xylose - 10 ONPG - 11 Indole -

12 Urease + 13 V- P - 14 Citrate + 15 TDA - 16 Gelatin - 17 Malonate + 18 Inositol - 19 Sorbitol - 20 Rhamnose - 21 Sucrose - 22 Lactose - 23 Arabinose + 24 Adonitol - 25 Raffinose - 26 Salicin - 27 Arginine +

3.3 Screening of supernatant for antimicrobial compound production To check whether the antimicrobial compound produced by isolate Pj1 is an extracellular compound, the supernatant of Pj1 was tested for its antimicrobial activity by paper disc diffusion method. Zone of clearance of 22 and 30 mm against Staphylococcus aureus, 20 and 25 mm against EPEC K-11, 14 and 20 mm

against Candida albicans, 7,7 mm and 8,29 mm against MRSA.

3.4 Identification of bacterial strains

Isolate Pj1 had a wide antimicrobial spectrum to all the test microorganisms pathogen. The 16S

rDNA sequence analysis indicated that Pj1 was most closely related to the member of the genus Pseudomonas aeruginosa strain ZC6. The highest sequence similarity values 99% and Genbank accession number KJ184550 (Figure 1)

Figure 1. Phylogenetic analysis of partial 16S rDNA gene sequences of bacteria isolate by employing bootstrap method.

4. Discussion Marine environment provide compound of rich diversity like bacteria associated with ascidian has been shown to have significant bioactivity like antimicrobial compound, isolate Pj1 from

Manokwari sea coast was performed in this study. Based on analysis of phylogenetic bacteria associated with ascidians included in the phylum Proteobacteria, class Gammaproteobacteria. Isolate Pj1 was phylogenetically related to

Pseudomonas aeruginosa strain ZC6 species with 99% identity in the 16S rDNA gene sequence. P aeruginosa will find anywhere including soil, water and plants. This bacteria often isolate from contaminated place and can producing metabolite like alginate, rhamnolipid, and pyocyanin (10). Pyocyanin is a kind of phenazine pigment which is soluble in water, chloroform and n-butanol. This pigment have antimicrobial activity against a variety of microorganisms. the product has strong toxicity for a wide range of organisms but the toxicity can be eliminated with adding superoxide dismutase (SOD) to the culture (5). The result from this study indicate the potential of bacterial to produce antimicrobial compound like pyocyanin and can be using for many applications and must be better exploration.

5. Acknowledgements

Authors are thankful to Directorate General of Higher Education, Ministry of Education and Culture, Indonesia to support this research; and the authors would like to thank Adhikari Dinesh, Ph.D candidate at Laboratory of Bioengineering, Faculty of Life Science, Ritsumeikan University for identification of this bacteria using 16S rDNA.

Conflict of interest statement

We declare that we have no conlict of interest References

(1) Amstrong, E., Liming. Y., Kenneth. G.B., Phillip. C.W., Grant, B. The Symbiotic role of Marine on living surfaces. Hydrobiologia . 2001. 461:37-40.

(2) Aneiros, A and Garateix, A. Bioactive peptides from marine sources: pharmacological properties and isolation procedures. Journal of Chromatography. 2004.803, 41-53.

(3) Cappucino, G.J and Sherman, N. Microbiology (A Laboratory Manual). Cummings Publishing Company Inc. 2001. New York. p 1-466.

(4) El-Shouny, W.A., Al-baidani, A.R.H & Hamza, W.T. Antimicrobial Activity of Pyocyanin Produce by Pseudomonas aeruginosa Isolated from Surgical Wound-Infections. International Journal of Pharmacy and Medical Sciences .2011. (1):01-07.

(5) Hasan, H.M & Fridovich, I. Mechanism of the antibiotic action of pyocyanine. Journal

Bacteriology. 1980. 141:156-163

(6) Imamura, N., Nishijima, M., Takadera, T., Adachi, K. New Anticancer Antibiotics Pelagiomicin Produced by a New Marine Bacterium Pelagiobacter variabilis. The Journal Antibiotic. 1997. 50(1)8-12.

(7) James, S.G., Holmstrom, C., Kjelleberg, S. Purification and Characterization of a Novel Antibacterial Protein from the Marine Bacterium D2. Applied and Environmental Microbiology. 1996. 62(8):2783-2788.

(8) Martinez-Garcia, M., Diaz-Valdes, M., Ramos-Espla, A., Salvador, N., Lopez, P., Larriba, E., Anton J. Cytotoxicity of the Ascidian Cystodytes dellechiajei Against Tumor Cells and Study of the Involvement of Associated Microbiota in the Production of Cytotoxic Compounds. Marine Drugs. 2007. 5: 52-70.

(9) Mearns-Spragg, A., Bregu, M., Boyd, K.G., Burgess, J.G. Cross-species Induction and Enhancement of Antimicrobial Activity Produced by Epibiotic Bacteria from Marine Algae and Invertebrates, after

Exposure to Terrestrial Bacteria. Letter Applied Microbiology. 1998. 27:142-146.

(10) Onsbasli, D and Aslim, B. Determination of antimicrobial activity and production of some metabolite by Pseudomonas aeruginosa B1 and B2 in sugar beet molasses. African Journal of Biotechology. 2008. vol 7 (24), 4614-4619.

(11) Penesyan, A., Maarshall-Jones, Z., Holmstrom, C., Kjelleberg, S., & Egan, S. Antimicrobial activity observed among cultured marine epiphytic bacteria reflects their potential as a source of new drugs. FEMS Microbiol Ecol. 2009. 69:113-124.

(12) Penesyan, A., Kjelleberg, S and Egan, S. Development of Novel Drugs from Marine Surface Associated Microorganisme. Marine Drugs. 2010. 8:438-459.

b. Resume kegiatan analisis Laboratorium dan data

a. Data Analisi Laboratorium . Analisis laboratorium meliputi : - Ekstrak genom DNA sampel bakteri dengan elektroforesis - Jumlah DNA bakteri dan jumlah sel bakteri dalam kultur - PCR dan sekuensing

Contoh hasil analisis laboratorium:

Hasil elektroforesis DNA bakteri

Hasil PCR DNA bakteri

- Jumlah DNA bakteri dan jumlah sel bakteri dalam kultur Di bawah in adalah data hasil sekuensing DNA bakteri berupa nukleotida

>Isolat1 AGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTTTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGTTTACAAAAGA

Total amount Amount of eDNA amount number number x 108

of eDNA (µl) with TE DNA in gel (ng) (µg mL1) cell ml/L

S.1 4 9 257.3 2.0584 349928000 3.50

S.2 4 9 192.6 1.5408 261936000 2.62

S.3 4 9 120.5 0.964 163880000 1.64

S.4 4 9 176.6 1.4128 240176000 2.40

S.5 4 9 204.8 1.6384 278528000 2.79

S.6 4 9 91.19 0.72952 124018400 1.24

S.7 4 9 161.2 1.2896 219232000 2.19

S.9 4 9 39.09 0.31272 53162400 0.53

S.12 4 9 47.06 0.37648 64001600 0.64

S.13 4 9 239.2 1.9136 325312000 3.25

S.14 4 9 42.74 0.34192 58126400 0.58

S.15 4 9 48.41 0.38728 65837600 0.66

S.16 4 9 170.9 1.3672 232424000 2.32

S.17 4 9 29.38 0.23504 39956800 0.40

S.18 4 9 61.25 0.49 83300000 0.83

Determine the number of marine bacteria

Volum TE bufferSample

>isolate 2 TGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGC

>isolate 4 GGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATG

CCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGC

>isolate 5 GGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCTCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATCAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAACAAACACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCTAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCTCCGGGCTCAACCTGGGAACTGCATTCGAGACTGGCTAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGCTGAAATGCCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGG

>isolate 6 TGCAGTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGC

GCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTA >isolate 7 TGCAAGTCGAGCGGATGAAGGGAGCTTGCTCCTGGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTGAGGGAGAAAGTGGGGGATCTTCGGACCTCACGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCAACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCAGCAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTACTGAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAAGCAAACAGGATTAGATACCCTGGTAGTGCACGCCGTAAACGATGTCGACTAGCCGTTGGGATCCTTGAGATATTAGTGGCGCAGCTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTAGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTTGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGCCTAACCGCAAGGGGGACG

>isolate 9 GTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCACACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAATGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTGGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAACCCTAGAGATAGGGCTTTTCC

TTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATTTTAGTTGCCATCATTTAGTTGGGCACTTTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATTTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGTTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCC

>isolate 12 GTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGACACACGGCCCACACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTGGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAACCCTAGAGATAGGGCTTTTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATTTTAGTTGCCATCATTTAGTTGGGCACTTTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATTTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGC >isolate 13 AGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAACGTACCTTTTGCTACGGAATAACTCAGGGAAACTTGTGCTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGCAAAGGATCGGCCCGCGTTGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGACTTTTAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGGAAGTCTTGAGTATGGTAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGACCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTTGGAGCAAACAGGATTAGATACCCTGG

TAGTCCACGCCGTAAACGATGAATGTTAGCCGTTGGGGAGTTTACTCTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATACCGGTCGCGGACACAGAGATGTGTCTTTCAGTTCGGCTGGACCGGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGCGCTGTGC

>isolate 14 AGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAACCCTAGAGATAGGGCTTTTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTTTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATTTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTGGAGCCAGCCGC

>isolate 15 GCAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTAT

TGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGAAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAAGGAGCTA

>isolate 16 GTCGAACGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAG

>isolate 17 AGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCA

ACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCC

>isolate 18 GCGACGGGCTACCTTATGTCTCGTCTCGGGGTAGCCATAGACAGCAGCTTGCTGCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAGACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGCTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCATGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTAGCAGAGATGCTTTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGTATACAAAAGAAGTAGGGAGCTATACCTACGGGGGGGCGACACAGAGAGTTGACTCACC

Pohon filogenik isolate bakteri yang diperoleh berdasarkan sekuensing 16S rDNA menggunakan maximum likehood

c. Rincian kegiatan selama sandwich

Time schedule for sandwich program in Japan

Month/Week Activities Notes I/1

I/2

- Consultations with supervisor in japan

- Discussion and orientation - Literature study - Discussion with research and

fellow doctoral candidates in Laboratory

October 2013

II/1-3 II/4

- Laboratory activities (Phylogenetic analysis of bacteria with antimicrobial activities from Indonesia ascidian)

- Discussions with Professor - Writing progress report

November 2013

III/1-2 III/3-4

- Discussion with research and fellow doctoral candidates

- Follow international conference as oral presentation in Kumamoto

- Laboratory activities (analysis compound bioactive from bacteria)

- Study academic writing - Presentation in laboratorium

group bioengineering - Preparations for return to Jakarta - (Indonesia)

December 2013

2. Surat-Surat Pendukung Dari Perguruan Tinggi/ Institusi Riset Di Luar Negeri a. Fotokopi LoA b. Fotokopi Rekomendasi dari pendamping di luar negeri c. Fotokopi naskah MOU d. Fotokopi sertifikat seminar di luar negeri e. Surat keterangan dari KBRI setempat

Graduate School of Life Sciences

1-1-1 Noji Higashi, Kusatsu, Shiga 525-8577 Japan

Declaration of Understanding

In receiving research supervision at the Graduate School of Life Sciences, Ritsumeikan

University (hereinafter refereed to as “RU”), I hereby declare that I understand and will

comply with the following in carrying out my research with integrity.

1. I will follow the instructions of my supervisor at RU, and comply with all rules and

regulations of RU.

2. I understand that if I engage in actions/behaviors that violate any of the rules and

regulations during my period of study abroad, I will lose my status as “Research

Student” at RU, and my research supervision will be terminated.

3. I agree that any intellectual property rights that arise through research supervision

provided by RU shall reside with RU.

4. Of the knowledge, experience, materials and information obtained through my

research activities, I will strictly keep secret those that are considered confidential

administrative or research information of RU. However, this does not include

anything that was already publicly available or that which can be proven to have

been in my possession; that which becomes publicly available in the future through

no breach of my own; or that which has been disclosed to me without obligation of

confidentiality by a third party who is lawfully entitled to make such disclosure.

5. I will not hold responsible the Sending Institution or RU for any damages received

by me during the period of study abroad at RU and will not make liability claims to

either party, unless the Sending Institution and/or RU is/are liable on grounds of

willful intent or gross negligence.

3. Curuculum Vitae

A. PERSONAL DATA: 1. NAMA : RINA ANITA MOGEA 2. GENDER : FEMALE 3. PLACE & DATE OF BIRTH : MANADO, 13 AUGUST 1973 4. HOME ADDRES : MT HARYONO 88 DINOYO

MALANG, PHONE 081344761562 5. EMAIL ADDRESS : [email protected]

6. MARITAL STATUS : SINGLE 7. EMPLOYMENT : BIOLOGY FACULTY

MATHEMATIC & SCIENCE PAPUA UNIVERSITY

8. CIVIL SERVICE DATA : NIP 197308132006042001 9. RANK : III C B. EDUCATION BACKGROUND: NO NAME OF

INSTITUTION YEAR

ATTENDED DEGREE/

CERTIFICATE FIELD OF STUDY

FROM TO

1 ELEMENTARY SCHOOL (RK II SANTA THERESIA MANADO)

1980 1986 CERTIFICATE

2. JUNIOR HIGH SCHOOL (SMP N 4 MANADO)

1986 1989 CERTIFICATE

3. HIGH SCHOOL (SMA N 1 MANADO)

1989 1992 CERTIFICATE BIOLOGY

4. SAMRATULANGI UNIVERSITY MANADO

1993 1992 DEGREE MARINE SCIENCE

5. SAMRATULANGI UNIVERSITY MANADO

2002 2005 MASTER ENVIRONMENT TOXICOLOGY

C. ACADEMIC BACKGROUND Scientific Publications Published In Journals: NO YEAR ARTICLE TITLE JOURNAL’S NAME

AND PLACE OF PUBLICATION

1. 2007 The Coastal Quality in Bunaken Island Observed by Condition of

Bacteriology

Jurnal Natural FMIPA-UNIPA

2. 2009 Bioecology of Sea Cucumber in Manokwari Water

Jurnal Perikanan dan Kelautan FPPK-UNIPA VOL 5 NO 1 MEI 2009

3. 2010 A Bioactive Compound of Lactarane Sesquiterpene Velleral From the

Stem Bark of Drymis beccariana

Gibbs.(Winteraceae)

The Future of Medicinal Plants. Surabaya ISBN 978-602-96839-1-2

4. 2010 Aspects of marine pollution based that bacteria coliform and

identification species in Doreri Bay Manokwari

Jurnal Ilmu Kelautan Undip Vol 15 no 1 maret

2010

MALANG, 22th MAY 2014 RINA ANITA MOGEA

I. Foto-Foto eksperimen di laboratorium

a . pemindahan isolat bakteri yang di bawah dari Indonesia

b. pemotongan pita DNA untuk pemurnian

Foto-foto Kampus

a

b

C

Foto-foto kampus : a dan b. foto di dalam laboratorium bioengenering Fakultas Mipa Universitas Ritsumeikan; c. Foto di luar laboratorium.

Foto-foto Walcome Party dengan mahasiswa baru dari Fakultas Mipa Universitas Ritsumeikan

Foto Farewell Party dengan para anggota lab bioengenering