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Ernawati Puji Rahayu
Evaluasi DNA untuk Mendeteksi Produk Olahan Daging yang Diduga Mengandung Bahan yang Bersumber dari Babi dengan Metode Taqman Real Time PCR = DNA Evaluation to Detect Processed Meat Products Suspected of Containing Ingredients Sourced from Pork Using the Taqman qPCR Method
"Metode Taqman MGB real time PCR yang cepat merupakan kunci pengawasan pemalsuan daging yang efektif. Penelitian bertujuan mengevaluasi kuantitas, kualitas DNA produk olahan daging babi, serta kandungan DNA babi produk olahan daging sapi yang diduga mengandung babi menggunakan Taqman MGB real time PCR untuk memverifikasi label. Lima produk olahan daging babi, 30 produk olahan daging sapi: dendeng, abon, baso, dan daging asap sebagai sampel, serta daging babi segar sebagai kontrol positif diekstraksi, diukur konsentrasi, kemurnian DNA, dielektroforesis serta diamplifikasi dengan realtime PCR. Konsentrasi, kemurnian DNA, nilai Ct sampel diuji ANAVA satu arah dilanjutkan uji Tukey, kecuali nilai Ct produk olahan daging sapi. Integritas DNA genomnya dianalisis deskriptif. Hasil uji ANAVA menunjukkan ada pengaruh nyata (P˂0,05) konsentrasi, kemurnian DNA dan nilai Ct. Hasil uji Tukey produk olahan daging babi: ada beda nyata konsentrasi DNA sampel dan kontrol positif (P˂0,05), kecuali kornet (P˃0,05). Kemurnian DNA baso dan daging asap berbeda nyata (P˂0,05) dengan kontrol positif. Nilai Ct sampel dan kontrol positif berbeda nyata (P˂0,05), kecuali dendeng (P˃0,05). Hasil uji Tukey produk olahan daging sapi: konsentrasi DNA baso dan daging asap berbeda nyata (P<0,05) dengan kontrol positif, kemurnian DNA kornet berbeda nyata (P<0,05) dengan kontrol positif. Semua DNA genom sampel terfragmentasi ukuran terendahnya sekitar 250 bp dimiliki kornet dan abon. Produk olahan daging dapat meningkat kuantitas DNAnya dan menurun kualitas DNAnya tergantung pada suhu dan bahan tambahan yang diberikan. Tiga puluh produk olahan daging sapi tidak mengandung DNA babi menggunakan Taqman real time PCR yang sensitif dan cepat serta terverifikasi mematuhi peraturan label.
The fast Taqman MGB qPCR method is key to effective meat adulteration surveillance. This research aimed to evaluate the quantity, quality of DNA from processed pork products and the content of pork DNA in processed beef products suspected of containing pork DNA using the Taqman MGB qPCR to verify labels. Five processed pork products, 30 processed beef products: corned, jerky, shredded, meatballs, and smoked meat were used as samples as well as and fresh pork as a positive control were extracted, DNA concentration and purity were measured, electrophoresed, and amplified with qPCR. The DNA concentration, purity, and Ct value were tested by one-way ANOVA followed by the Tukey test, except for the Ct value of processed beef products. The genomic DNA integrity was analyzed descriptively. The ANOVA showed a significant effect (P˂0.05) on the concentration and purity of DNA and Ct value. Tukey test results for processed pork products: there was a significant difference (P˂0.05) in the DNA concentration of the samples and positive controls, except for corned (P˃0.05). The DNA purity of pork meatballs and smoked pork was significantly different (P˂0.05) from the positive control. The Ct values of the samples and positive control were significantly different (P˂0.05), except for jerky (P˃0.05). The results of the Tukey test for processed beef products: the DNA concentration of beef meatballs and smoked beef was significantly different (P<0.05) with the positive control, and the DNA purity of corned beef was significantly different (P<0,05) with positive control. All genomic DNA samples were fragmented with the smallest size of about 250 bp experienced by corned and shredded. Processed meat products can increase the quantity of DNA and decrease the quality depending on temperature and additives. Thirty processed beef products did not contain pork DNA using the sensitive and fast Taqman qPCR and verified to comply with label regulations."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2022
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UI - Tesis Membership  Universitas Indonesia Library
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Dewi Ayu Sekarini
Analisis Lokasi Mikroplastik Pada Insang Ikan Bandeng Chanos chanos (Forsskal, 1775) Dari Tambak Marunda = Analysis of Microplastic Location of Milkfish Gilss Chanos chanos (Forsskal, 1775) From Marunda Pond
"Penelitian ini menganalisis jumlah, jenis, dan ukuran mikrplastik yang tersangkut pada lembar insang ikan bandeng yang dibudidaya di Tambak Marunda, Jakarta Utara. Kriteria pengambilan sampel ikan bandeng adalah yang berumur sekitar 5—6 bulan (siap konsumsi) sebanyak 10 ekor. Organ yang diteliti adalah insang ikan bandeng pada lenbar terluar (anterior hemibranch), tengah, dan dalam (posterior hemibranch). Masing-masing lembar tersebut dibagi menjadi tiga bagian yaitu atas (upper), tengah, dan bawah (lower). Insang ikan bandeng diisolasi dari organ tubuh lainnya kemudian dibagi berdasarkan posisi lembar insang. Sampel insang kemudian diamati dengan object glass yang ditetesi akuades di bawah mikroskop cahaya dan didokumentasikan menggunakan kamera. Ukuran mikroplastik dianalisis menggunakan aplikasi ImageJ. Jenis mikroplastik yang ditemukan adalah fiber, film, fragmen, dan granula dengan persentase fiber 66%, film 14%, granula 14%, dan fragmen 6%. Ukuran partikel fiber memiliki rentang 974—5000 µm, film 144—628,8 µm, fragmen 157—1.125 µm, dan granula 117,7—484 µm. Komposisi mikroplastik terbanyak ditemukan di lembar terluar (6.667 partikel) > dalam (6.480 partikel) > tengah (6.449) dan komposisi pada sisi kanan insang lebih tinggi (10.414 partikel) dibandingkan sisi kiri insang (10.055 partikel).
This research analyzed the number, types, and size of microplastics in milkfish gills sheets from cultivated pond in Marunda, North Jakarta. The criteria for the sample were fish that ready for consumption (around 5—6 month old) and 10 individuals. The organs studied were the outer sheet (anterior hemibranch), middle (posterior hemibranch), and inner (posterior hemibranch) of milkfish gills on each sheet was divided into three parts, namely upper, middle, and lower. The gills were isolated from other organs and then divided based on position of the gills sheets. The gills were then placed on object glass dripped with distilled water under a light microscope and were documented using a camera device. Microplastic size was analyzed using ImageJ (Application). Microplastic types found include fiber, film, fragment, and granule with 66% fiber, 14% film, 14% granule, and 6% fragment. The particle size of the fiber has a range of 974—5000 µm, film 144—628,8 µm, fragment 157—1.125 µm, and granule 117,7—484 µm. The highest microplastic composition was found in outer sheet (6,667 particles) > inner (6,480 particles) > middle (6,449 particles) and the composition on the right side of the gills was higher (!0,414 particles) than the left side of the gills (10,055 particles)."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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UI - Skripsi Membership  Universitas Indonesia Library
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Andika Satria Putra Prabaswary
Pemberian laktosa pada visualisasi inti oosit domba Garut (ovis aries l.) setelah maturasi dan pascakriopreservasi = Administration of lactose for visualization of sheep oocyte core after maturation and postcryopreservation.
"Penelitian mengenai potensi laktosa terhadap peristiwa swollen inti oosit domba garut (Ovis aries L.) pascamaturasi dan pascakriopreservasi telah dilakukan. Tujuan penelitian ini adalah menganalisis kemampuan laktosa serta mencari konsentrasi terbaik dalam visualisasi inti oosit (swollen) domba garut (Ovis aries L.) setelah maturasi dan pascakriopreservasi. Sebanyak 143 oosit yang memiliki kualitas A dan B (sitoplasma homogen, zona pelusida utuh, dan lapisan kumulus lebih dari 4 lapis) dimaturasi dalam medium TCM-199 dengan penambahan Bovine Serum Albumin (BSA). Oosit dengan status inti mencapai Metafase II (M-II) hasil maturasi in vitro tersebut, kemudian diberikan perlakuan laktosa berbagai konsentrasi untuk dilihat peristiwa swollen intinya. Perlakuan laktosa juga diberikan kepada oosit pascakriopreservasi dengan memperhatikan kondisi metabolisme oosit yang masih normal dan viabel. Swollen inti oosit diamati menggunakan pewarna Hoechst & PI di bawah mikroskop fluoresens. Hasil penelitian menunjukkan bahwa adanya perbedaan antarkonsentrasi secara uji Kruskal-Wallis (P > 0,05) dan konsentrasi laktosa 3% menunjukkan persentase peristiwa swollen inti oosit pada oosit pascamaturasi yang tertinggi (58,22%) dan juga pada oosit pascakriopreservasi (48,83%). Peristiwa swollen inti oosit pascamaturasi pada konsentrasi laktosa 3% memiliki perbedaan nyata dibandingkan konsentrasi laktosa 0%, 1%, dan 5%. Peristiwa swollen inti oosit pascakriopreservasi pada konsentrasi laktosa 3% tidak ada perbedaan nyata yang signifikan dengan konsentrasi laktosa 0%, 1%, dan 5%. Laktosa memiliki kemampuan untuk swollen inti oosit domba garut dan konsentrasi laktosa 3% merupakan konsentrasi terbaik yang mampu menyebabkan peristiwa swollen inti oosit domba garut pascamaturasi dan pascakriopreservasi.
Research on the potential of lactose against post-maturation and postcryopreservation events of oocyte core swollen of garut sheep (Ovis aries L.) has been carried out. The purpose of this study was to analyze the ability of lactose and to find the best concentration in visualizing the oocyte core (swollen) of garut sheep (Ovis aries L.) after maturation and post-cryopreservation. A total of 143 oocytes with A and B qualities (homogeneous cytoplasm, intact zona pellucida, and a cumulus layer of more than 4 layers) were saturated in TCM-199 medium with the addition of Bovine Serum Albumin (BSA). Oocytes with core status reached Metaphase II (M-II) as a result of in vitro maturation, then treated with various concentrations of lactose to see the core swollen event. Lactose treatment was also given to post-cryopreservation oocytes by taking into account the normal and viable conditions of oocyte metabolism. Oocyte core swollen was observed using Hoechst & PI stain under fluorescence microscope. The results showed that the difference between concentrations by Kruskal-Wallis test (P> 0.05) and 3% lactose concentration showed that the highest percentage of oocyte nucleus swollen events in post-maturation oocytes (58.22%) and also in post-cryopreservation oocytes (48.83 %). Post-maturation oocyte nucleus swollen events at 3% lactose concentration had a significant difference compared to 0%, 1%, and 5% lactose concentrations. The post-cryopreservation of oocyte core swollen at 3% lactose concentration was no significant difference with 0%, 1%, and 5% lactose concentrations. Lactose has the ability to swollen the nucleus of garut sheep oocytes and the 3% lactose concentration is the best concentration capable of causing postmaturation and post-cryopreservation of arrowroot oocyte core swollen events."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2021
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UI - Skripsi Membership  Universitas Indonesia Library
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Indah Tama Arina
Maskulinisasi ikan cupang koi (betta splendens) fancy menggunakan hormon 17α-metiltestosteron melalui perendaman larva selama 24 jam = Masculinization of betta koi fish (betta splendens) fancy using hormone 17α-methyltestosterone through soaking larvae for 24 hours
"Ikan cupang (Betta splendens) fancy berkelamin jantan merupakan salah satu jenis ikan yang digemari oleh masyarakat. Ikan cupang berkelamin jantan memiliki keunggulan pada bentuk dan warnanya. Upaya untuk memperoleh populasi jantan dapat dilakukan dengan cara pengalihan kelamin dengan perendaman hormon 17α-metiltestosteron. Penelitian ini bertujuan untuk mengetahui pengaruh hormon 17α-metiltestosteron dengan konsentrasi berbeda 1, 2, 3 mg/L, serta kontrol melalui perendaman larva umur 5 hari dalam hormon 17α-metiltestosteron selama 24 jam. Penelitian ini dilakukan dengan menggunakan metode eksperimental Rancangan Acak Lengkap (RAL) dengan empat perlakuan dosis hormon 17α-metiltestosteron (1,2,3 mg/L, dan kontrol) dengan masing-masing enam ulangan setiap perlakuan. Ikan yang telah berumur 76 hari setelah perendaman hormon diidentifikasi jenis kelaminnya. Data yang diperoleh diuji dengan uji normalitas, uji homogenitas, uji ANAVA satu faktor, dan uji Tukey. Hasil analisis data berdasarkan data morfologi menunjukkan bahwa baik nilai kelulushidupan ikan maupun persentase jantan terdistribusi normal dan homogen. Hasil uji ANAVA satu faktor dengan tingkat kepercayaan 99% menunjukkan ada perbedaan nyata (P<0,01) antara perlakuan hormon dengan perlakuan kontrol terhadap kelulushidupan maupun persentase pengalihan kelamin menjadi jantan (maskulinisasi). Kelulushidupan ikan cupang koi (Betta splendens) fancy tertinggi ditunjukkan pada perlakuan kontrol yaitu sebesar 48,97%, sedangkan persentase jantan (maskulinasi) tertinggi 56,84% ditunjukkan pada perlakuan 17α-metiltestosteron 2 mg/L.
Male Betta fish (Betta splendens) fancy is one of the types of fish favored by the community. Effort to obtain male population can be done by sex reversal through immersion of the hormone 17α-methyltestosterone. This study aims to determine the effect of 17α-methyltestosterone hormone through immersion of 5-day-old-larvae hormone for 24 hours. This research was conducted using a completely randomized experimental design with four treatments of the 17α-methyltestosterone hormone dosage (1, 2, 3 mg/L, and control) with six replications for each treatment. Fishes that were 76 days old after hormone immersion were identified each of sex based on its morphology. The data obtained were tested by normality test, homogeneity test, ANOVA test, and Tukey test. The results of data analysis based on morphological data show that both the survival value of fish and the percentage of males are normally distributed and homogeneous. The results of ANOVA of one factor with a 99% confidence level showed that there was a significant difference (P<0,01) between hormone treatment with control treatment on survival and the percentage of sex reversal into males. The highest survival rate of male Betta fish (Betta splendens) fancy 48,97% was shown by control treatment, while the highest percentage of male 56,84% was shown by 2 mg/L."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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UI - Skripsi Membership  Universitas Indonesia Library
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Ananda Febriyani
Kualitas Semen Beku Kambing Saanen (Capra aegagrus hircus) pada Pengencer Semen berbasis Lesitin dan Liposom dengan Penambahan Maltosa = The Quality of Saanen Goat (Capra aegagrus hircus) Frozen Semen in Lecithin and Liposomes-based Semen Extender with Addition of Maltose
"Kambing Saanen merupakan salah satu kambing perah penghasil susu yang baik. Guna meningkatkan efisiensi reproduksi kambing Saanen, dikembangkan teknologi inseminasi buatan (IB). Salah satu faktor pendukung keberhasilan IB yaitu tersedianya semen beku sesuai kriteria. Tujuan dari penelitian yaitu mengetahui pengencer semen yang optimal untuk semen beku kambing Saanen dengan membandingkan pengencer semen berbasis lesitin (Andromed®) dan liposom (Optixcell®) dengan penambahan maltosa 0,4%. Metode koleksi semen dilakukan dengan menggunakan vagina buatan. Semen kambing Saanen kemudian diperiksa secara makroskopis dan mikroskopis. Teknik penyimpanan semen beku dengan teknik kriopreservasi pada suhu -196ºC selama 10 menit. Evaluasi kualitas semen meliputi motilitas, viabilitas, dan membran plasma utuh (MPU). Hasil yang diperoleh diuji secara statistik dengan uji ANAVA satu faktor kemudian uji Tukey. Hasil penelitian menunjukkan tidak terdapat perbedaan nyata (P>0,05) pada nilai rata-rata persentase motilitas perlakuan Andromed® (50,83±3,76); Andromed® + Maltosa 0,4% (50,83±3,76); Optixcell® (50,83±3,76); Optixcell® + Maltosa 0,4% (48,33±6,06), nilai rata-rata persentase viabilitas Andromed® (54,67±3,50); Andromed® + Maltosa 0,4% (54,50±2,51); Optixcell® (56,50±3,45); Optixcell® + Maltosa 0,4%(52,83±5,78), dan nilai rata-rata persentase MPU spermatozoa Andromed® (56,00±3,80); Andromed® + Maltosa 0,4% (56,50±5,47); Optixcell® (53,83±5,31); Optixcell® + Maltosa 0,4% (53,33±6,06). Hasil penelitian menyimpulkan bahwa pengencer semen liposom menghasilkan kualitas yang sama baik dengan pengencer semen berbasis lesitin dan penambahan maltosa 0,4% pada pengencer semen berbasis liposom dan lesitin tidak berpengaruh terhadap kualitas semen beku kambing Saanen yang dihasilkan
The Saanen goat is a notable domestic goat that performs nicely in dairy production, such as milk. Efforts to improve the reproduction efficiency of Saanen goats, include the development of artificial insemination (IB) technology. A significant factor that effect AI is the availability of frozen semen according to satisfactory criteria. This research aims to determine the optimal semen extender for Saanen goat frozen semen by comparing lecithin‒based extender (Andromed) and liposomes (Optixcell) when added 0.4% maltose. Semen collection utilises artificial vagina, then is examined macroscopically and microscopically. The semen storage technique used in this study was the cryopreservation technique at -196 for 10 minutes. Criteria of semen quality includes motility, viability, and intact plasma membrane. The result obtained were statistically tested with the ANAVA one way factor test then Tukey test. The result showed that there was no significant difference (P>0.05) in the average value of the motility percentage of treatments Andromed (50.83±3.76); Andromed + Maltosa 0.4% (50.83±3.76); Optixcell (50.83±3.76); Optixcell + Maltosa 0.4% (48.33±6.06), viability Andromed (54.67±3.50); Andromed + Maltosa 0.4% (54.50±2.51); Optixcell (56.50±3.45); Optixcell + Maltosa 0.4% (52.83±5.78), and intact plasma membrane Andromed® (56.00±3.80); Andromed + Maltosa 0.4% (56.50±5.47); Optixcell (53.83±5.31); Optixcell + Maltosa 0.4% (53.33±6.06). The results of the study concluded that the liposomes extender produced the same quality as the lecithin-based extender and the addition of 0.4% maltose in liposomes and lecithin‒based extender had no effect on the quality of the frozen semen Saanen goat."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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UI - Skripsi Membership  Universitas Indonesia Library
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Bimayu Rati
Pengaruh Penambahan Maltosa ke dalam Pengencer Semen Berbasis Liposom terhadap Kualitas Semen Beku Kambing Saanen (Capra aegagrus hircus) = Effect of Maltose Addition to Liposome Based Extender on Quality Saanen Goat Frozen Semen (Capra aegagrus hircus)
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Kambing Saanen merupakan kambing perah yang dapat menghasilkan susu mencapai 2.695,3 kg dalam satu masa laktasi. Salah satu upaya untuk meningkatkan efisiensi reproduksi serta meningkatkan kualitas kambing perah yaitu penerapan teknik Inseminasi Buatan (IB). Dalam proses pembekuan semen untuk inseminasi buatan, pengenceran semen dan pemberian krioprotektan berpengaruh terhadap kualitas semen beku yang dihasilkan. Penelitian ini bertujuan untuk mengetahui pengaruh penambahan maltosa dengan berbagai konsentrasi ke dalam pengencer berbasis liposom (Optixcell®) terhadap kualitas spermatozoa kambing Saanen selama kriopreservasi. Penampungan semen dilakukan dengan metode vagina buatan, lalu dilanjutkan dengan pemeriksaan makroskopis dan mikroskopis. Selanjutnya, semen diproses menjadi semen beku menggunakan nitrogen cair -196°C dan setiap tahap kriopreservasi dilakukan evaluasi secara mikroskopis dengan parameter motilitas, viabilitas, dan membran plasma utuh. Penelitian menggunakan Rancangan Acak Lengkap (RAL) dengan empat perlakuan dan enam ulangan. Empat perlakuan terdiri dari kontrol (OK); Optixcell®+Maltosa 0,2% (O2); Optixcell®+Maltosa 0,4% (O4); Optixcell®+Maltosa 0,6% (O6). Data penelitian diuji menggunakan Analisis Varian (ANAVA) satu faktor. Hasil penelitian menunjukkan tidak adanya perbedaan nyata (P>0,05) pada nilai rata-rata presentase motilitas, viabilitas, dan membran plasma utuh. Penambahan maltosa ke dalam pengencer berbasis liposom (Optixcell®) tidak memiliki pengaruh dalam mempertahankan kualitas semen beku kambing Saanen.

 

Saanen goat is a dairy goat that can produce milk up to 2,695.3 kg in one lactation period. One of the way to improve reproductive efficiency and quality of dairy goat by implementing Artificial Insemination (IB) techniques. During the process of freezing semen for artificial insemination, the extender and cryoprotectant agents affect the quality of frozen semen produced. The aim of this study was to examine the effect of maltose addition into liposome based extender (Optixcell®) on quality of Saanen goat during cryopreservation. Semen was collected using artificial vagina, then immediately evaluated in macroscopic and microscopic. Furthermore, the semen was processed into a frozen semen using liquid nitrogen -196°C and every cryopreservation steps were evaluated microscopically using sperm quality parameters such as motility, viability, and membrane integrity. The experimental design was RAL with four treatments and six repetition. Four treatments consisted of Controls (K); Optixcell®+Maltose 0,2% (O2); Optixcell®+Maltose 0,4% (O4); Optixcell®+Maltose 0,6% (O6). The data were analyzed by one way ANAVA. In conclusion, there was no significant difference (P>0.05) in average percentage of motility, viability, and membrane integrity. Addition of maltose to liposome based extender (Optixcell®) has no significantly to maintain the quality of frozen semen Saanen goat.

 

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Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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UI - Skripsi Membership  Universitas Indonesia Library
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Vallery Athalia Priyanka
Transformasi plasmid rekombinan yang termetilasi sebagai pembawa kaset gen protease untuk proses integrasi kromosomal ke bacillus halodurans CM1 = Transformation of methylated plasmid as protease gene carriers for the chromosomal integration into bacillus halodurans CM1
"Efisiensi transformasi plasmid rekombinanyang rendah ke dalam bakteri wild-type disebabkan oleh mekanisme pertahanan dari sel bakteri. Enzim restriksi dalam sel bakteri target dapat mendegradasi plasmid rekombinan. Transformasi plasmid pBBRE194 rekombinan yang mengandung gen protease dari Bacillus halodurans CM1 (pBBRE194 prot-CM1) telah dilakukan, tetapi efisiensi transformasi rendah dan klona rekombinan yang didapatkan tidak menunjukkan sifat yang stabil. Penelitian ini bertujuan untuk memetilasi plasmid pBBRE194 prot-CM1 dan transformasi plasmid pBBRE194 prot-CM1 termetilasi secara konjugasi ke B. halodurans CM1, kemudian menganalisis aktivitas enzim protease yang dihasilkan B. halodurans CM1 pembawa plasmid pBBRE194 prot-CM1. Aktivitas spesifik (U/mg) protease yang dihasilkan oleh B. halodurans CM1 rekombinan dianalisis dengan cara mengukur unit aktivitas (U/mL) dengan metode Amano dan kadar protein (mg/mL) dengan metode Bradford. Plasmid pBBRE194 prot-CM1 dalam E. coli TOP10 berhasil dimetilasi oleh gen methylase pada plasmid pPAMC125 yang diinduksi oleh 0,02% L-arabinosa. Konjugasi plasmid pBBRE194 prot-CM1 termetilasi ke B. halodurans CM1 berhasil dilakukan dan terpilih 1 klona B. halodurans CM1 rekombinan yang telah terverifikasi. Verifikasi dilakukan berdasarkan kemampuan klona dalam mendegradasi protein pada media selektif yang mengandung skim milk dan tetracycline. Verifikasi berdasarkan polymerase chain reaction dengan mendeteksi sekuens gen resistan tetracycline pada klona juga dilakukan. Proses PCR koloni pada B. halodurans CM1 rekombinan menunjukkan terbentuknya pita DNA ukuran 1024 bp yaitu ukuran gen resistan tetracycline pada plasmid pBBRE194 prot-CM1. Hasil analisis menunjukkan bahwa aktivitas spesifik B. halodurans CM1 rekombinan (660,700 U/mg) lebih rendah dibandingkan dengan kontrol negatif, yaitu B. halodurans CM1 wild-type (1054,928 U/mg). Analisis aktivitas protease dilakukan pada suhu 50 oC dan pH 12.
Transformation rate into wild-type bacteria is commonly low because of the cell defense mechanism of the bacteria. Restriction modification (RM) in bacteria cells can prevent the introduction of recombinant plasmid into target bacteria. Previously, the transformation of recombinant shuttle vector pBBRE194 containing protease gene (pBBRE194 prot-CM1) into wild-type Bacillus halodurans CM1 has been conducted. However, the transformation rate seemed low, and the stable recombinant clones could not be obtained. Therefore, in vivo methylation of this plasmid in E. coli has to be done before genetic transformation into the wild-type bacterium, to obtain stable recombinant B. halodurans CM1. In this study, a plasmid with artificial modification (pPAMC125) harboring genes encoding for the modification enzymes (methylases) from another strain, B. halodurans C-125, and pBBRE194 prot-CM1 plasmid were transformed by conjugation into B. halodurans CM1. Specific activity (U/mg) of protease produced by recombinant B. halodurans CM1 was analyzed by measuring activity units (U/mL) by the Amano method and protein quantity (mg/mL) by the Bradford method. The pBBRE194 prot-CM1 might be methylated by methylases that was induced by 0.02% L-arabinose. Conjugation of the methylated pBBRE194 prot-CM1 to B. halodurans CM1 was successfully carried out and recombinant B. halodurans CM1 was verified. The verification of a recombinant clone is based on its ability to degrade proteins on selective media containing skim milk and tetracycline. Also beside, verification based on polymerase chain reaction was also carried out by detecting tetracycline resistance gene sequences. The PCR result in recombinant clone amplified the 1024 bp DNA, which the size of the tetracycline resistance gene in pBBRE194 prot-CM1 plasmid. The analysis of protease activity showed that the specific activity of the recombinant clone (660.700 U/mg) was lower than the negative control, which B. halodurans CM1 wild-type (1054.928 U/mg). The analysis was carried out at 50oC and pH 12. "
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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UI - Tesis Membership  Universitas Indonesia Library
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Widamayanti
Isolasi gen fungsional resistan eritromisin dan kanamisin dari bacillus halodurans CM1 = Isolation of erythromycin and kanamycin resistance functional gene from bacillus halodurans CM1
"Bacillus halodurans CM1 berpotensi sebagai inang dalam menghasilkan beberapa jenis enzim yang berguna, seperti xilanase, lipase, dan protease. Selain sebagai penghasil enzim, salah satu gen potensial lainnya adalah gen resistan antibiotik. Penelitian ini bertujuan untuk mengetahui kemampuan resistansi B. halodurans CM1 terhadap eritromisin dan kanamisin serta diperoleh produk gen fungsional resistan eritromisin dan kanamisin dari B. halodurans CM1. Isolasi gen eritromisin dan kanamisin dari B. halodurans CM1 belum pernah dilakukan. Berdasarkan uji KHM, B. halodurans CM1 resistan terhadap eritromisin dan kanamisin. Isolasi gen resistan eritromisin dan kanamisin dilakukan dengan menggunakan metode amplifikasi PCR. Produk PCR yang diduga gen resistan eritromisin, yaitu gen ErmK dan BH0381. Produk PCR yang diduga gen resistan kanamisin, yaitu gen aadK dan APH. Gen ErmK dikloning dengan menggunakan kloning vektor pGEM-T Easy, sedangkan gen BH0381, aadK, dan APH dikloning dengan menggunakan kloning vektor pJET1.2/blunting. Vektor rekombinan ditransformasi ke Escherichia coli DH5alfa. Hasil analisis sekuens DNA menggunakan BLAST menunjukkan bahwa gen ErmKCM1 dan BH0381 masing-masing memiliki kemiripan 99,65% (GenBank No access: BH0380, ErmK) dan 99,45% (GenBank No access: BH0381, mphB) dengan sekuens dari B. halodurans C-125. Sekuens gen ErmKCM1 dan BH0381CM1 menunjukkan bahwa dua gen tersebut merupakan gen yang fungsional. Sekuens upstream dari gen BH0381CM1 dianalisis dan diperoleh bahwa terdapat 50 pb yang diduga promoter. Hasil analisis sekuens DNA menggunakan BLAST menunjukkan bahwa gen aadKCM1 dan APHCM1 masing-masing memiliki kemiripan 97,73% (GenBank No access: BH0322) dan 99,47% (GenBank No access: BH0326) dengan sekuens dari B. halodurans C-125. Hasil penyejajaran gen aadKCM1 menunjukkan adanya delesi enam pasang nukleotida pada lokus ke-354 hingga 359 yang menyebabkan frameshift, sehingga gen aadKCM1 tidak memiliki sekuens yang open reading frame (ORF). Hasil translasi gen aadKCM1 dan APHCM1 menunjukkan bahwa hanya sekuens gen APHCM1 dapat ditranslasi menjadi protein yang fungsional. Hasil uji resistansi kanamisin pada transforman yang membawa plasmid rekombinan promoter gen APHCM1-ORF dapat menyandikan resistan kanamisin dengan konsentrasi kanamisin sebesar 20 µg/mL.
Bacillus halodurans CM1 has potential as a host in producing several types of useful enzymes, such as xylanase, lipase, and protease. Besides industrial enzymes, one of the other potential genes is the antibiotic-resistant gene. This study aims to determine the ability of B. halodurans CM1 resistance to erythromycin and kanamycin and to obtain erythromycin and kanamycin-resistant functional gene products from B. halodurans CM1. Isolation of erythromycin and kanamycin genes from B. halodurans CM1 has never been done. Based on the MIC test, B. halodurans CM1 is resistant to erythromycin and kanamycin. Erythromycin and kanamycin resistance gene isolation was carried out using PCR amplification method. PCR products suspected of being erythromycin resistance genes, namely ErmK and BH0381 genes. PCR products suspected of being kanamycin resistance genes, namely genes aadK and APH. The ErmK gene was cloned using the pGEM-T Easy vector cloning, while the BH0381, aadK, and APH genes were cloned using the pJET1.2/blunting vector cloning. The recombinant vector was transformed into Escherichia coli DH5ï?¡. The results of DNA sequence analysis using BLAST showed that the ErmKCM1 and BH0381 genes each had 99.65% similarities (GenBank No access: BH0380, ErmK) and 99.45% (GenBank No access: BH0381, mphB) with the sequence of B. halodurans C-125. The results of the translation of the ErmKCM1 and BH0381CM1 genes indicate that the two genes are functional genes. The upstream sequence of the BH0381CM1 gene was analyzed and it was found that 50 bp was suspected as a promoter. The results of DNA sequence analysis using BLAST showed that the aadKCM1 and APHCM1 genes each had a similarity of 97.73% (GenBank No access: BH0322) and 99.47% (GenBank No access: BH0326) with the sequence of B. halodurans C-125. The alignment of the aadKCM1 gene shows the deletion of six nucleotide pairs at the 354-359 locus that causes frameshift, so the aadKCM1 gene does not have an open reading frame (ORF) sequence. The translation of aadKCM1 and APHCM1 genes show that only the APHCM1 gene sequence can be translated into a functional protein. The results of kanamycin resistance test on transformants carrying plasmids with native promoter APHCM1-ORF gene can encode kanamycin resistance with a kanamycin concentration of 20 µg/mL."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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Rizka Arifah Iskandar
Identifikasi mutasi gen MLH1 dan MSH2 pada pasein kanker kolon menggunakan teknik Multiplex Ligation-Dependent Probe Amplification (MLPA) = Identification of MLH1 and MSH2 gene mutation in colon cancer patients using Multiplex Ligation-Dependent Probe Amplification (MLPA)
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Gen mismatch repair (MMR) merupakan gen yang berperan dalam mekanisme DNA repair. Gen MMR dapat memperbaiki kesalahan pasangan basa, perubahan nukleotida dan kesalahan dalam unit pengulangan pasangan basa (microsatellites) selama proses replikasi DNA. Mutasi pada MMR yang umum terjadi pada gen MLH1 dan MSH2 memiliki asosiasi dengan terjadinya Lynch Syndrome pada pasien kanker kolon. Lynch Syndrome (LS) atau Hereditary Non-Polyposis Colorectal Carcinoma (HNPCC) merupakan sindrom kanker kolon herediter yang paling umum diwariskan. LS dapat ditandai dengan mutasi gen MLH1 dan MSH2 berupa delesi dan duplikasi large genomic. Multiplex ligation-dependent probe amplification (MLPA) merupakan teknik kuantifikasi berbasis PCR yang dapat digunakan untuk mendeteksi perubahan copy number variant pada large genomic. Tujuan dari penelitian ini adalah untuk mengidentifikasi mutasi gen MLH1 dan MSH2 pada kanker kolon dengan teknik Multiplex Ligation-Dependent Probe Amplification (MLPA). Sebanyak 25 sampel darah pasien yang terdiagnosis kanker kolon yang dikumpulkan secara retrospektif di Rumah Sakit Kanker Dharmais dikoleksi dari tahun 2018-2019. Mutasi gen MLH1 dan MSH diidentifikasi pada sampel darah pasien yang terdiagnosis kanker kolon dengan teknik MLPA menggunakan Probemix P003-D1 MLH1/MSH2 (MRC-Holland). Penelitian ini berhasil melakukan optimalisasi penggunaan teknik MLPA dengan terpenuhinya kualitas fragmen kontrol MLPA, kualitas fragment analysis, dan comparative analysis pada perangkat lunak Coffalayser.Net. Hasil screening pada 25 sampel menunjukkan tidak ditemukan adanya pola mutasi gen MLH1 dan MSH2 pada sampel yang mengindikasikan tidak terjadinya Lynch Syndrome dan kanker kolon yang terjadi bersifat sporadis.

Mismatch repair genes (MMR) plays a role in the mechanism of DNA repair. The MMR genes correct base pair errors, nucleotide changes, and errors in base pair repeating units (microsatellites) during DNA replication. Mutations in MMR are common in MLH1 and MSH2 genes that have an association with the occurrence of Lynch Syndrome in colon cancer. Lynch Syndrome or Hereditary Non-Polyposis Colorectal Carcinoma (HNPCC) is the most common inherited hereditary colon cancer syndrome. LS is characterized by MLH1 and MSH2 gene mutations in the form of large genomic deletions and duplication. Multiplex ligation-dependent probe amplification (MLPA) is a PCRbased quantification technique that can detect changes in copy number variants in large genomics. The aim of the study was to identify MLH1 and MSH2 gene mutations in colon cancer with Multiplex Ligation-Dependent Probe Amplification (MLPA) technique. Twenty-five blood samples of patients diagnosed with colon cancer were collected in Dharmais Cancer Hospital collected in 2018-2019, retrospectively. Mutations in MLH1 and MSH2 genes were identified in blood samples of patients diagnosed with colon cancer by the MLPA technique using Probemix P003-D1 MLH1/MSH2 (MRC-Holland). This study succeeded in optimizing the use of MLPA technique by fulfilling the fragments quality control of MLPA, the quality of fragment analysis, and comparative analysis in Coffalayser.Net software. The results of 25 samples screening showed no pattern of MLH1 and MSH2 gene mutations in the sample. This data indicated that there was no Lynch Syndrome and the colon cancer was sporadic.

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Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2020
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UI - Skripsi Membership  Universitas Indonesia Library
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Jocelyn Nataniel
Optimasi Ekspresi Gen Sintetik Penyandi Enzim Pendegradasi Plastik Polietilena Tereftalat (PETase) pada Escherichia coli Arctic Express (DE3) = Optimization of Polyethylene Terephthalate Plastic Degrading Enzyme (PETase) Synthetic Coding Gene Expression in Escherichia coli Arctic Express (DE3)
"Masalah limbah plastik polietilena tereftalat (PET) di Indonesia menjadi perhatian yang serius karena penguraiannya lambat dan berpotensi merusak lingkungan. Solusi yang menjanjikan adalah Ideonella sakaiensis polyethylene terephthalate hydrolase (IsPETase) yang dapat mendegradasi plastik dengan lebih cepat. IsPETase sebelumnya telah diekspresikan di Escherichia coli BL21 (DE3). Namun, IsPETase masih terekspresikan secara insoluble sehingga IsPETase perlu diamati ekspresi gen dan optimasi kondisi ekspresi pada E. coli Arctic Express (DE3). Penelitian bertujuan untuk mengamati ekspresi gen PETase pada E. coli Arctic Express (DE3) dan menentukan kondisi optimal untuk ekspresi. Penelitian ini melibatkan berbagai tahapan seperti peremajaan kultur rekombinan, produksi protein, dan pemanenan, yang dioptimalkan untuk kondisi ekspresi. Ekspresi gen PETase diamati pada fraksi ekstraseluler (dipekatkan), periplasmik (dipekatkan), dan sitoplasmik. Fraksi ekstraseluler belum terekspresikan secara optimal sehingga optimasi kondisi ekspresi dilanjutkan pada fraksi sitoplasmik (soluble) dengan media pertumbuhan Luria Bertani (LB), induksi IPTG 1,0 mM selama 8 jam, dan waktu sonikasi selama 10 menit menghasilkan aktivitas spesifik PETase 0,07 U/mg. Namun, pemurnian protein dan ekspresi perlu dilakukan dengan sel inang Bacillus.
The problem of polyethylene terephthalate (PET) plastic waste in Indonesia has become a serious concern due to its slow degradation and potential environmental damage. A promising solution is Ideonella sakaiensis polyethylene terephthalate hydrolase (IsPETase), which can degrade plastic faster. IsPETase has been expressed in Escherichia coli BL21 (DE3), but it is still expressed insolubly, so the expression of the gene and the optimization of the expression conditions in Escherichia coli Arctic Express (DE3) need to be observed. This study aims to observe the PETase gene expression in E. coli Arctic Express (DE3) and determine the optimal conditions for expression. This study involves various stages, such as refreshing culture, protein production, and harvesting, which are optimized for expression conditions. PETase gene expression was observed in the extracellular (concentrated), periplasmic (concentrated), and cytoplasmic soluble fractions. The extracellular fraction has not been optimally expressed, so expression optimization continued in the cytoplasmic fraction with Luria Bertani (LB) growth medium, IPTG induction of 1.0 mM for 8 hours, and sonication time for 10 minutes, resulting in specific activity of 0.07 U/mg. However, protein purification is required and expression is performed with Bacillus host cells."
Depok: Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Indonesia, 2023
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UI - Skripsi Membership  Universitas Indonesia Library
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