Dasar-dasar Kimia Hayati (KI-2261) (bagian 2)

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

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

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

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

9 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

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

11 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

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

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

14 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

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 5.2 Pemurnian bagian-bagian sel/organel 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
Figure 5-24 Copyright (c) by W. H. Freeman and Company

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

19 5.4 Struktur sel tanaman Figure 5-43
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 5.4 Organel-organel sel eukaryot 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 5.4 Lysosomes 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 5.4 Peroxisomes 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 5.4 Mitochondria 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 5.4 Chloroplasts Figure 5-46 Copyright (c) by W. H. Freeman and Company

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 5.4 The endoplasmic reticulum (ER) 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 5.4 The Golgi complex 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 5.4 The nucleus Copyright (c) by W. H. Freeman and Company Figure 5-50

28 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

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

30 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

31 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

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

33 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

34 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

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

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

37 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

38 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

39 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

40 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

41 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

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

43 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

44 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

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

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

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