2016년 1 학기 생명과학 및 실험기법 2 박 제 권 가천대학교 생명과학과

이론 및 실습 진행 계획 5월 10일 (1주차) : 조교 소개, 실험 주의사항 (실험복/안전사고/음식물반입 절대금지)및 이론 5월 17일(2주차) : 형질전환 대장균 배양(기본 4조건)/각 조별 LB, LB+Amp, LB+IPTG, LB+Amp+IPTG - 18일 (수요일) – 대표 –원심분리후 상층액 냉동보관 5월 24일 (3주차) – 단백질 정량 및 Dot-blot.

Induction & confirmation Gene Over-expression Induction & confirmation

Genome Project 유전자지도 및 물리 지도를 제작하여 질병관련 유전자 의 위치를 확인. 생물 정보학 (Bio-informatics)및 비교 유전체학 (Comparative genomics) 등을 통하여 인간 진화의 원리 를 이해. 특히, 인간 genome이 암호화 하는 모든 유전자 및 그 조절 기전을 파악, 보다 효과적인 생명공학 기법을 개발.

출아형 효모 (budding yeast, S. cerevisiae): 진핵 생물 중 가장 먼저 게놈 프로젝트가 완성됨

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유전자의 조작 Biological manipulation of the cell and genes Gene Cloning 특정 조각 DNA의 분리 Recombinant DNA Technology 시험관 안에서 새로운 DNA 분자 구축 Genetic Engineering In vitro culture Stem cell line Transgenesis (형질 전환) 살아있는 생물체에 재도입 -cells -animals 궁극적인 목표: 세포 –환경- 이웃 세포들과 상호작용 기전을 이해

10_03_Cells_origin.jpg in vitro culture 세포의 작용 기전을 연구하기 위해서는 그 구성 요소 - 생화학적으로 분석- 이를 위해서는 각 세포군마다 1)순수하게 분리 된 2)많은 숫자의 세포가 필요. in vitro culture cell sorting (cluster of differentiation markers) 10_03_Cells_origin.jpg

Steps of Recombinant DNA Technology (유전자 재조합 기술) gene cloning -cDNA library를 screen 한다. -PCR cloning Construction of recombinant vector (plasmid? Cosmid? phagemid?) Bacterial Transformation Genetic Manipulation -mutagenesis -promoter fusion -reporter construct Transformed cells and animals

Cloning 의 목적 제한된 양의 시발물질(목적의 DNA)로부터 많은 양의 재조합 DNA 분 자를 생산할 수 있다. ng의 DNA → ug(1,000배) → mg(100만 배)의 재조 합 DNA 획득). 유전자의 구조, 기능, 발현 등에 이용. 정제된 재조합 DNA 분자만을 얻을 수 있음. 결합되지 못한 운반체 분자 제거. 결합하지 못한 DNA 단편 제거(exonuclease). Recycling 된 운반체 DNA의 제거. 원치 않은 DNA 단편이 결합된 재조합 DNA. 정확한 재조합 DNA만을 획득. *** Cloning: 재조합 DNA 분자를 만드는 것 만을 의미하는 것이 아니라, 재조합 DNA 분자를 숙주세포에 도입하는 형질전환 까지를 의미함.

Transfection 방법 Chemical Method. Calcium phosphate 이용법. Liposome 이용법. Physical Method. Electroporation. Microinjection. Virus 이용법. Retro virus 이용 Adeno virus 이용. Adeno associate virus 이용.

형질전환 발전 단계 1970. Mandel and Higa. 형질전활 율 증가. Ice-cold CaCl2 solution(⋋ phage). 1972. Cohen et al. plasmid DNA transformation 성공. 1978. Kushner. 2가 양이온(+ 2)과 DMSO 이용. 1983. Hanahan. Hexamine cobalt chloride.

Competent cell 이란? 정상적인 세균세포(Bacterial cell)에 화학적 처리를 하여 외래 DNA가 세균세포(숙주세포)에 잘 들어가게 제조된 세 포(형질전환 허용 세포 또는 수용성 세포). 화학적 처리에 사용하는 시약. Calcium chloride, Hexamine cobalt chloride, Manganase chloride, Dimethyl sulfoxide(DMSO). Calcium chloride가 가장 많이 이용 – 이미 완성된 Plasmid 움를 형질전환 할 경우.. Electroporation (신속하고 매우 고효율적 방법->라이브러 리 스크리닝 할 때 주로 사용) Amp sensitive(Amp-), Tet sensitive(Tet-).

세균 이외의 형질전환 효모(Yeast): Saccharomyces cerevisiae. 식물세포 동물세포 Lithidium chloride. Lithidium acetate. 식물세포 Cell wall 분해(Cellulase, Chitinase). Electroporation 이용. 동물세포 Manipurator(Microinjection) 이용. Calcium chloride 이용.

by Complementation (shot gun) Gene Cloning by Library Screening by PCR by Complementation (shot gun) Genomic vs. cDNA library

cDNA library = A still shot of a busy cross section 10_25_cDNA.jpg

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c Why construct a recombinant plasmid? stable propagation in bacteria cDNA Why construct a recombinant plasmid? stable propagation in bacteria separation Why transform bacteria? stable storage amplification 10_23_genomic.library.jpg

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10_24_hybridization.jpg Colony Hybridization 10_24_hybridization.jpg

Gene Cloning by Library Screening Gene Cloning by PCR Gene Cloning by Complementation

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Recombinant DNA Technology (보다 큰 효과를 가진 효소, 독성 없는 백신, etc.) 2) 인위적으로 돌연변이를 유도-> 해당 유전자의 기능 파악 2) 특이적 활성물질의 대량 생산 (가격 인하) 3) 유전자 발현의 시기, 특정위치(장소) 조사 (in situ hybridization, reporter gene) 4) 형질전환 동물 및 세포의 제작 (유용한 물질 생산, 질병 모델, 줄기세포 치료 등…) 5) 법의학에 응용…어떻게?

EXP.2. PCR analysis of clone # 9 using set of primers. Template: purified pTa+Va7.3 +RFP (by using Qiagen). Set Primers: Va 7.3 –F & R RFP – F & R Va7.3-F & RFP- F Va7.3-F & RFP-R. 1 2 3 4 F Va7.3 RFP R pTa-cassette *** Finally… cloned pTa+Va7.3+RFP. For the Tg exp., need to isolate more pTa+Va7.3+RFP…

OOOOOOh, YES !!!! 1 2 Lane 1. un-cut 2. // XmaI / NotI RFP, ~700bp Verification of both Va7.3 and RFP insertion into pTa-cassette vector Purified pTa+Va7.3+RFP (in frame) briefly; / by Xma I, at 37C, O/N - E-OH precipitation, at -80C for 30 min. / by Not I, at 37 C, 2 hrs pTa-cassette Va7.3, ~400bp RFP, ~800 bp Xma I Not I Not I 1 2 Lane 1. un-cut 2. // XmaI / NotI RFP, ~700bp Va7.3, ~400bp OOOOOOh, YES !!!!

Genetic Engineering

10_04_Restrict.nuclease.jpg 1) Cutting and Joining DNA fragment

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10_05_gel.electrophor.jpg 2) Hybridization (혼성화)

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4) Microarray 10_15_DNA.microarrays .jpg 10_15_DNA.microarrays .jpg

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SDS-PAGE & Western blotting

SDS-PAGE purposes To separate protein molecules on the basis of molecular weight May then be electroblotted for immunoanalysis. To determine the molecular weights of unknowns by comparison to standards

HRP + ECL substrate  light HRP conjugated 2o antibody 1o antibody Proteins blotted to membrane from SDS-PAGE gel What’s wrong? Membrane

SDS-PAGE preparation of cell extract Lyse cells in RIPA buffer containing inhibitors of both proteases and phosphatases. Centrifuge lysate to remove membranous cellular debris. Determine protein concentration in g/l of the lysate. Example: Bradford method - Binding of Coomassie Blue dye by proteins in solution and comparison to standards of known concentration

preparation of cell extract components of the lysis buffer 50 mM Tris-HCl, pH 8.0 – pH friendly to most proteins 150 mM NaCl, isotonic saline Detergents – disrupt lipid bilayers; aid in solubilizing hydrophobic proteins NP-40 (non-ionic, good solubilization, weakly denaturing) Deoxycholate (a bile acid, ionic, moderately denaturing) SDS (synthetic, ionic, excellent solubilization, strongly denaturing)

Components of the lysis buffer (cont’d) Dithiothreitol – a reducing agent Prevents inappropriate oxidation of reduced cysteines to disulfide bonds, and thereby Prevents covalent aggregation and precipitation of proteins that are not covalently linked in vivo. DTT is especially important for native gel electrophoresis. (See Slide #12. DTT in the lysis buffer is insufficiently concentrated to disrupt the disulfide bonds which form the natural structure of some proteins, but is sufficiently concentrated to prevent inappropriate disulfide bonds from forming.)

Components of the lysis buffer (cont’d) Protease inhibitors Prevent protein degradation and thereby allow more accurate determination of molecular weight Examples PMSF – inhibits serine proteases phenylmethanesulfonylfluoride Leupeptins tripeptides produced by various species of Actinomycetes L-leucyl-L-leucyl-D-argininal modified at NH –terminus by acetyl or propionyl Aprotinin Found in pancreas and lung, among other tissues Natural inhibitor of various extra- and intracellular proteases

Components of the lysis buffer (cont’d) Phosphatase inhibitors Inhibitors prevent enzymatic removal of phosphates from phosphorylated proteins during extract preparation. Phosphorylated and dephosphorylated proteins migrate differently during SDS-PAGE. Useful information can be gained by knowing whether or not a protein is phosphorylated in vivo in given cells under specific conditions. Examples of general phosphatase inhibitors NaF Na3VO4

SDS-PAGE preparation of sample for loading Major components of the sample loading “buffer” SDS DTT Tracking dye Glycerol

SDS-PAGE preparation of sample for loading Major purposes of boiling in loading buffer are to denature and coat proteins with SDS All proteins bind SDS with similar ratios of detergent to protein mass. (-) charge on dodecyl sulfate ions  ~ = charge/mass ratio for all proteins, so separation is on the basis of size. reduce disulfide bonds using DTT (or  -mercaptoethanol) Causes disulfide bonded peptides to become independent (see next slide. Good for determining size of disulfide-bonded subunits

2 2 H H Heat Excess DTT  + H

Reduction by monovalent mercaptans -mercaptoethanol Reduction by divalent mercaptans dithiothreitol (reduced) dithiothreitol (oxidized) DTT dithiothreitol

preparation of sample for loading Dye is included to monitor migration during PAGE Bromphenol blue Glycerol is included to make sample denser than running buffer minimizes diffusion during loading

SDS-PAGE gel system (Note features in red!) Discontinuous Two gel layers with different polyacrylamide concentrations A different buffer for each of the two parts of the gel And a third buffer as the running buffer Stacking (concentrating) gel 4% acrylamide (36.5:1, acryl/bis) 125 mM Tris-H+Cl-, pH 6.8, 0.1% SDS Resolving (separating) gel 10% acrylamide (36.5:1, acryl/bis) 425 mM Tris-H+Cl-, pH 8.8, 0.1% SDS Running buffer 25 mM Tris base; 192 mM glycine, pH 8.3; 1% SDS

Why use a discontinuous gel and buffer system for SDS-PAGE? Purpose of the stacking gel: to concentrate all the proteins in the sample into a thin band at the top of the resolving gel Makes it possible to use a dilute sample Purpose of the resolving gel: to separate the proteins on the basis of size. The next set of slides will address how the stack works. Following that will be a set of slides on the resolving system.

stacking gel Concentrates proteins because it Has large pores (4%), so proteins of all sizes move easily through the pores of the stacking gel until they meet a frictional barrier at the top of the resolving gel, with its smaller pores (10%). But that’s not the only way the proteins are concentrated! Clever design of a discontinuous buffer system increases the concentrating effect of the stacking gel on the proteins in the sample!

stacking gel Concentrates proteins also because it uses the stacking and running buffers to form a voltage gradient   protein mobility Stacking gel buffer is of Low salt concentration Cl- = leading ion because it is small, negatively charged, and moves quickly through gel Running buffer ion is primarily glycine = trailing “ion”, which at pH 6.8 is nearly neutral A region of low ionic strength quickly develops between Cl- and glycine, generating a voltage gradient. Large, negatively charged proteins are left to constitute most of the molecular current, and move quickly to the bottom of the stacking gel.

pH 6.8, neutral Notice the progression of Cl-, negatively charged proteins of different sizes (P-), and mostly neutral glycine (G) in the next three slides.

P-- P-- P- P- P- P- G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

P-- P-- P- P- P- P- G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

P-- P-- P- P- P- P- G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

P-- P-- P- P- P- P- G G G G G G G Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl- Cl-

On to the resolving gel . . . The resolving gel separates proteins as a function of percentage acrylamide ratio of acrylamide to bis extent of difference in size between the proteins being resolved

The resolving gel Has a higher [ion] (0.425 M Tris-H+Cl-) than the stacking gel (0.125 M Tris-H+Cl-) so Proteins contribute less to the total ionic current than they did in the stacking gel, and as a result the mobilities of proteins  and differences in mobilities among proteins of different sizes become more apparent.

Resolving gel In addition, the resolving gel has a higher pH (8.8) than the stacking gel (6.8) so glycine takes on a more negative charge, thereby the increasing the total ion concentration and because it is small, moves ahead of the proteins. So proteins move slowly through the gel and are resolved by friction on the basis of size.

H2 pH 6.8, neutral pH 8.8, negatively charged

Myosin, 202 kd MWs adjusted to account for masses of covalently bound dyes -galactosidase, 133 kd BSA, 71 kd Carbonic anhydrase, 41.8 kd Soybean trypsin inhibitor, 30.6 kd Lysozyme, 17.8 kd Aprotinin, 6.9 kd

Sample of SDS- PAGE

Protein gel (SDS-PAGE) that has been stained with Coomassie Blue.

What happens after electrophoresis? 1. Fix the proteins in the gel and them stain them. 2. Electrophorectic transfer to a membrane and then probe with antibodies- (Western blotting) (Refer Western Blot first few slides)

Western Blot Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.

…Western Blot Proteins are separated by gel electrophoresis, usually SDS- PAGE. The proteins are transferred to a sheet of special blotting paper called nitrocellulose. The proteins retain the same pattern of separation they had on the gel.

..Western Blot The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

..Western Blot The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

..Western Blot

..Western blotting Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. This antibody is used as a probe to detect the protein of interest.

Western Blot followed by SDS Proteins are separated using SDS-polyacrylamide gel electrophoresis which separates proteins by size. Nitrocellulose membrane is placed on the gel. The actual blotting process may be active (electroblotting) or passive (capillary). Electroblotter is used for faster and more efficient transfer of protein from gel to membrane Sandwich of filter paper, gel, membrane and more filter paper is prepared in a cassette, which is placed between platinum electrodes. An electric current is passed through the gel causing the proteins to electrophorese out of the gel and onto the nitorcellulose membrane.

Terminologies.. The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA. Southwestern blotting, based along the lines of Southern blotting (which was created by Edwin Southern) and first described by B. Bowen and colleagues in 1980, is a lab technique which involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA).

Terminologies.. Dot blot a mixture containing the molecule to be detected is applied directly on a membrane as a dot. Protein detection using the dot blot protocol is similar to western blotting in that both methods allow for the identification and analysis of proteins of interest.

References Introduction to Biotechnology by W.J. Thieman and M.A. Palladino. Pearson & Benjamin Cummings 2nd edition. http://www.toodoc.com/SDS-PAGE-ppt.html http://www.bio.davidson.edu/courses/genomics/method/ Westernblot.html http://en.wikipedia.org/wiki/Dot_blot