Dasar-dasar Kimia Hayati (KI-2261) (bagian 2) F. Warganegara 4/2/2017 KI-1213-2

Topik Bahasan Teknik- Teknik Analisis Sel: Pengantar : Tipe dan Jenis Sel: Prokarya ke Eukarya Uni - Multi-selular Molekul, Energi dan Metabolisme: Komponen Kimia Sel Energi dan keteraturan Biologi Struktur, Bentuk dan Informasi makromolekul struktur dan fungsi asam Nukleat Struktur protein Protein sebagai biokatalis Teknik- Teknik Analisis Sel: Struktur sel dalam mikroskop Isolasi dan penumbuh- an sel Fraksinasi dan Analisis Komponen Sel Sel dalam Bioteknologi modern 4/2/2017 KI-1213-2

Teknik- Teknik Analisis Sel 4/2/2017 KI-1213-2

5 Struktur Sel di bawah Mikroskop Mikroskopi: Magnification, Contrast, Resolution Light Microscopy (200 nm) Brightfield Fluorescent Advanced Electron Microscopy (1 nm) Transmission Scanning Copyright (c) by W. H. Freeman and Company

5.1 Microskop cahaya

5.1.1 Brightfield microscopy Masalah: Sel umumnya tak berwarna & transparan Agar strutkturnya terlihat  perlu diwarnai Dapat melekat dan mudah dibuat irisan yang sangat tipis Masalah baru yang timbul akibat tindakan di atas: Mengubah struktur sel/molekul Hanya memberikan gambaran “sel mati” Copyright (c) by W. H. Freeman and Company

5.1.2 Fluorescent microscopy Memungkinkan lokalisasi molekul sel spesifik Pewarna Fluorescent “berpendar” di dalam gelap Zat warna tsb dapat langsung atau tidak langsung berasosiasi dengan molekul sel: Eg: Rhodamin (merah) Fluorescein (hijau) Beberapa pewarna fluoresen dapat digunakan secara berturutan Dengan mikroskop ini dapat ditampilkan sel mati atau hidup Copyright (c) by W. H. Freeman and Company

Bagan alat Mikroskop fluoresen Aktin dari kultur sel fibroblast Figure 5-5 Copyright (c) by W. H. Freeman and Company Figure 5-6

Mikroskopi cahaya untuk objek 3-D Confocal Scanning or Deconvolution Microscopy Generates 3D images of living cells Removes out-of-focus images  optical sectioning Can look inside thick specimens (eggs, embryos, tissues) Figure 5-9 A mitotic ferilized egg of sea urchin (Psammechinus): a) fluorescent microscopy, b) confocal microscopic image Copyright (c) by W. H. Freeman and Company

5.1.3 Advanced light microscopy Memungkinkan pengamatan sel hidup yang transparan Pergeseran fasa cahaya (Light phase shifts) yang disebabkan oleh specimen digunakan untuk menciptakan contrast Phase contrast (refracted and unrefracted light) Differential interference contrast (two light beams) Figure 5-14 Time-lapse micrographs of cultured fibroblast cell movement along a glass surface Copyright (c) by W. H. Freeman and Company

5.2 Mikroskop Elektron 5.2.1 Transmission electron microscopy (TEM) Operates in vacuum Specimen usually fixed, embedded, sectioned, and stained with an electron-dense material Special techniques: Metal shadowing: visualize surface structures, cell components Cryoelectron: visualize unfixed, unstained samples Freeze fracture, freeze etch: visualize membrane interior Freeze etch: visualize cell interior Copyright (c) by W. H. Freeman and Company

Bagan alat Transmission electron microscope Copyright (c) by W. H. Freeman and Company Figure 5-15 Figure 5-16

5.2.2 Scanning electron microscopy Can visualize surfaces of tissues, cells, isolated cell parts Specimen is fixed and coated with thin layer of heavy metal Images secondary electrons, resolution = 10 nm Figure 5-20 Copyright (c) by W. H. Freeman and Company

5.3 Pemurnian sel dari campurannya dengan flow cytometry Membutuhkan penanda yang berfluorescent untuk sel target Figure 5-21 Copyright (c) by W. H. Freeman and Company

5.2 Pemurnian bagian-bagian sel/organel Understanding the roles of each each cell component depends on methods to break open (lyse) cells and separate cell components for analysis Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution Separation of cell components generally involves centrifugation 5.2 Pemurnian bagian-bagian sel/organel Copyright (c) by W. H. Freeman and Company

5.3.2 Faraksinasi sel dengan sentrifugasi differensial Copyright (c) by W. H. Freeman and Company Figure 5-23

5.3.3 Pemisahan Organel dengan sentrifugasi kesetimbangan gradien densitas Figure 5-24 Copyright (c) by W. H. Freeman and Company

5.4 Struktur sel hewan Figure 5-42 Copyright (c) by W. H. Freeman and Company

5.4 Struktur sel tanaman Figure 5-43 Copyright (c) by W. H. Freeman and Company Figure 5-43

5.4 Organel-organel sel eukaryot Lysosomes Peroxisomes Mitochondria Chloroplasts the Endoplasmic Reticulum (ER) the Golgi complex the Nucleus the Cytosol 5.4 Organel-organel sel eukaryot Copyright (c) by W. H. Freeman and Company

5.4 Lysosomes Responsible for degrading certain cell components material internalized from the extracellular environment Key Features single membrane pH of lumen  5 acid hydrolases carry out degradation reactions 5.4 Lysosomes Copyright (c) by W. H. Freeman and Company Figure 5-44a

5.4 Peroxisomes Responsible for degrading fatty acids toxic compounds Key Features single membrane contain oxidases and catalase 5.4 Peroxisomes Copyright (c) by W. H. Freeman and Company

5.4 Mitochondria Site of ATP production via aerobic metabolism Key Features outer membrane intermembrane space inner membrane matrix 5.4 Mitochondria Copyright (c) by W. H. Freeman and Company Figure 5-45

5.4 Chloroplasts Site of photosynthesis in plants and green algae Key Features outer membrane intermembrane space inner membrane stroma thylakoid membrane thylakoid lumen 5.4 Chloroplasts Figure 5-46 Copyright (c) by W. H. Freeman and Company

5.4 The endoplasmic reticulum (ER) Responsible for most lipid synthesis most membrane protein synthesis Ca++ ion storage detoxification Key Features network of interconnected closed membrane tubules and vesicles composed of smooth and rough regions 5.4 The endoplasmic reticulum (ER) Copyright (c) by W. H. Freeman and Company Figure 5-47

5.4 The Golgi complex Modifies and sorts most ER products Key Features series of flattened compartments & vesicles composed of 3 regions: cis (entry), medial, trans (exit) each region contains different set of modifying enzymes 5.4 The Golgi complex Copyright (c) by W. H. Freeman and Company Figure 5-49

5.4 The nucleus Separates DNA from cytosol transcription from translation Key Features outer membrane inner membrane nuclear pores nucleolus 5.4 The nucleus Copyright (c) by W. H. Freeman and Company Figure 5-50

The portion of the cell enclosed by the plasma membrane but not part of any organelle Key Features the cytoskeleton polyribosomes metabolic enzymes 5.4 The cytosol Copyright (c) by W. H. Freeman and Company Figure 5-52

Sel dalam Bioteknologi modern Kultur sel, bakteri dan virus 4/2/2017 KI-1213-2

6.1 Keunggulan bekerja dengan kutur sel dibandingkan dengan organisme utuh Lebih homogen dibaningkan dengan sel-sel dalam jaringan Dapat disesuaikan dengan kondisi percobaan Dapat mengisolasi sel tunggal dan ditumbukan menjadi koloni degan materi genetik yang seragam Copyright (c) by W. H. Freeman and Company

Pertumbuhan mikroba dalam kulturnya Contoh: E. coli dan yeast S. cerevisiae Memiliki pertumbuhan yang cepat dan hanya memerlukan nutrisi yang sederhana Dapat ditumbuhkan dalam agar semipadat Strain mutan dapat diisolasi dengan cara replica plating Yeast colonies Figure 6-1 Copyright (c) by W. H. Freeman and Company

6.1 Replica plating Figure 6-2 Copyright (c) by W. H. Freeman and Company

2 Pertumbuhan sel hewan dalam kulturnya Memerlukan media yang kaya, termasuk asam amino esensial, vitamin, garam, glukosa dan serum Kebanyakan hanya tumbuh dalam permukaan padat yang khusus A single mouse cell A colony of human cells Many colonies in a petri dish Figure 6-3 Copyright (c) by W. H. Freeman and Company

6.2 Sel Primer dan galur sel (Primary cells and cell lines) Kultur Sel Primer (Primary cell cultures) diperoleh dari jaringan hewan Tipe sel tertentu mudah dikulturkan, yang lain tidak Jika sel dipisahkan dari hewan utuhnya dan berhasil membelah diri, umumnya hanya sampai periode terbatas (sekitar 50 doublings), dan akhirnya mati Sel “transformant tertentu” dapat menjadi immortal dan dapat digunakan untuk membentuk galur sel (cell line) Transformant ini bisa berasal dari tumors atau timbul secara spontan Laju transformasi spontan bervariasi antar species Copyright (c) by W. H. Freeman and Company

6.2 Pembentukan kultur sel Figure 6-5 Copyright (c) by W. H. Freeman and Company

2 Beberapa kultur sel dapat berdeferensiasi membentuk struktur seperti jaringan Copyright (c) by W. H. Freeman and Company Figure 6-7b,c

6.2 Fusi Sel Dua sel yang berbeda dapat diinduksi untuk bergabung menghasilkan sel hibrida (hybrid cell, heterokaryon) Hibrida Interspesifik dapat digunakan untuk genetika sel somatik Sel hibrida tertentu (hybridomas) digunakan untuk produksi antibodi monoklonal Copyright (c) by W. H. Freeman and Company Figure 6-8

6.2 Produksi antibodi monoklonal terhadap protein X Figure 6-10 6.2 Produksi antibodi monoklonal terhadap protein X Copyright (c) by W. H. Freeman and Company

3 Viruses: struktur, fungsi, dan kegunaan Virus adalah parasit kecil yang tidak dapat bereproduksi sendiri virus menginfeksi sel tertentu dan menggunakan mesin reproduksi sel inang untuk memperbanyak virus Virus terdiri atas asam nukleat (RNA atau DNA) dikelilingi oleh lapisan/kulit protein Viruse dapat menginfeksi sel prokaryot atau eukaryot dan rentang sel yang dapat bertindak sebagai sel inang dari viruses terbatas (sempit) Study tentang virus memberikan pemahaman mengenai aspek dasar biologi sel dan pembentukan cancer Copyright (c) by W. H. Freeman and Company

6.3 Bentuk virus Figure 6-11 The protein coat (capsid) of a virus is constructed of multiple copies of a single or a few different proteins Some virus capsids are also surrounded by a envelope consisting of a lipid bilayer and a few glycoproteins Copyright (c) by W. H. Freeman and Company

6.3 The lytic replication cycle of E. coli bacteriophage T4 Early proteins replicate viral DNA and induce expression of late proteins Late proteins include capsid and assembly proteins and enzymes to degrade the host cell DNA Copyright (c) by W. H. Freeman and Company Figure 6-16

6.3 The lytic replication cycle of an enveloped virus Figure 6-17 Copyright (c) by W. H. Freeman and Company

6.3 Virus Bakteri yang sering digunakan pada penelitian biokimia dan genetika T phages of E. coli Temperate phages (bacteriophage ) Small DNA phages RNA phages Copyright (c) by W. H. Freeman and Company

6.3 Bacteriophage  undergoes either lytic replication or lysogeny following infection of E. coli Figure 6-19 Copyright (c) by W. H. Freeman and Company

6.3 Commonly used Class I, II, and III viruses Copyright (c) by W. H. Freeman and Company

6.3 Commonly used Class IV, V, and VI viruses Copyright (c) by W. H. Freeman and Company

6.3 The retroviral life cycle Figure 6-22 Copyright (c) by W. H. Freeman and Company