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Presentasi berjudul: "Dasar-dasar Kimia Hayati (KI-2261) (bagian 2) F. Warganegara 6/21/2014KI-1213-21."— Transcript presentasi:

1 Dasar-dasar Kimia Hayati (KI-2261) (bagian 2) F. Warganegara 6/21/2014KI

2 Topik Bahasan  Struktur protein  Protein sebagai biokatalis 5. Teknik- Teknik Analisis Sel:  Struktur sel dalam mikroskop  Isolasi dan penumbuh- an sel  Fraksinasi dan Analisis Komponen Sel  Sel dalam Bioteknologi modern 6/21/2014KI Pengantar : 2. Tipe dan Jenis Sel:  Prokarya ke Eukarya  Uni - Multi-selular 3. Molekul, Energi dan Metabolisme:  Komponen Kimia Sel  Energi dan keteraturan Biologi 4. Struktur, Bentuk dan Informasi makromolekul  struktur dan fungsi asam Nukleat

3 Teknik- Teknik Analisis Sel 6/21/2014KI

4 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 5.1 Microskop cahaya

6 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

7 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

8 Bagan alat Mikroskop fluoresen Figure 5-5 Figure 5-6 Aktin dari kultur sel fibroblast

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

10 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) Copyright (c) by W. H. Freeman and Company Figure 5-14 Time-lapse micrographs of cultured fibroblast cell movement along a glass surface

11 5.2 Mikroskop Elektron 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

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

13 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 Copyright (c) by W. H. Freeman and Company Figure 5-20

14 5.3 Pemurnian sel dari campurannya dengan flow cytometry Copyright (c) by W. H. Freeman and Company Figure 5-21 Membutuhkan penanda yang berfluorescent untuk sel target

15 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 Copyright (c) by W. H. Freeman and Company

16 5.3.2 Faraksinasi sel dengan sentrifugasi differensial Copyright (c) by W. H. Freeman and Company Figure 5-23

17 5.3.3 Pemisahan Organel dengan sentrifugasi kesetimbangan gradien densitas Copyright (c) by W. H. Freeman and Company Figure 5-24

18 5.4 Struktur sel hewan Copyright (c) by W. H. Freeman and Company Figure 5-42

19 5.4 Struktur sel tanaman Copyright (c) by W. H. Freeman and Company Figure 5-43

20 5.4 Organel-organel sel eukaryot  Lysosomes  Peroxisomes  Mitochondria  Chloroplasts  the Endoplasmic Reticulum (ER)  the Golgi complex  the Nucleus  the Cytosol Copyright (c) by W. H. Freeman and Company

21 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 Copyright (c) by W. H. Freeman and Company Figure 5-44a

22 5.4 Peroxisomes  Responsible for degrading  fatty acids  toxic compounds  Key Features ◦ single membrane ◦ contain oxidases and catalase Copyright (c) by W. H. Freeman and Company

23 5.4 Mitochondria  Site of ATP production via aerobic metabolism  Key Features ◦ outer membrane ◦ intermembrane space ◦ inner membrane ◦ matrix Copyright (c) by W. H. Freeman and Company Figure 5-45

24 5.4 Chloroplasts  Site of photosynthesis in plants and green algae  Key Features ◦ outer membrane ◦ intermembrane space ◦ inner membrane ◦ stroma ◦ thylakoid membrane ◦ thylakoid lumen Copyright (c) by W. H. Freeman and Company Figure 5-46

25 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 Copyright (c) by W. H. Freeman and Company Figure 5-47

26 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 Copyright (c) by W. H. Freeman and Company Figure 5-49

27 5.4 The nucleus  Separates ◦ DNA from cytosol ◦ transcription from translation  Key Features ◦ outer membrane ◦ inner membrane ◦ nuclear pores ◦ nucleolus Copyright (c) by W. H. Freeman and Company Figure 5-50

28 5.4 The cytosol  The portion of the cell enclosed by the plasma membrane but not part of any organelle  Key Features ◦ the cytoskeleton ◦ polyribosomes ◦ metabolic enzymes Copyright (c) by W. H. Freeman and Company Figure 5-52

29 Sel dalam Bioteknologi modern Kultur sel, bakteri dan virus 6/21/2014KI

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

31 Copyright (c) by W. H. Freeman and Company Pertumbuhan mikroba dalam kulturnya 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

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

33 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 Figure 6-3 A colony of human cellsMany colonies in a petri dish

34 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

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

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

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

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

39 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

40 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

41 Copyright (c) by W. H. Freeman and Company 6.3 The lytic replication cycle of E. coli bacteriophage T4 Figure 6-16 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

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

43 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

44 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

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

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

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


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