Transformation Biology experiment.

Presentation on theme: "Transformation Biology experiment."— Presentation transcript:

1 Transformation Biology experiment

2 Historical Perspective
Frederic Griffith 1928 London First controlled demonstration of genetic transformation Griffith made the observation that nonpathogenic bacteria (Streptococcus pneumoniae) became pathogenic when mixed with a virulent strain of heat-killed S. pneumoniae (i.e. injected mixture killed mice) Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 Oswald Avery demonstrated that DNA is responsible for conferring pathogenic properties

3 Transformation Transformation is a natural process that Bacterial have evolved in order to obtain DNA from their environment. Genetically modification of a cell Involves uptake of foreign DNA Replication within organism Gene expression DNA  RNA  Protein (central dogma) Cell DNA Term Prokaryote Plasmid Transformation Bacteriophage (virus) Infection Eukaryote Transfection Virus vector

4 Method of Transformation
Electroporation Electrical shock makes cell membranes permeable to DNA Microinjection Using a glass micropipette Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock

5 Plasmid Small circular dsDNA separate from bacterial DNA
Plasmids exist in bacteria, yeast Single or multiple plasmid copies per cell Easy to isolate and manipulate Used as vector for transforming bacteria with foreign DNA Foreign DNA is inserted after cutting with restriction enzymes Plasmids contain certain genes which offer a competitive advantage for bacteria (i.e. antibiotic resistance) Positive Selection: confers growth advantage i.e. able to grow in presence of antibiotic Insert gene for expression (<10kb insertion)

6 Plasmid 1) Replication origin plasmid가 자가복제할 때 처음 인식하는 부위
2) Antibiotics resistance gene 항생제에 내성을 나타내는 유전자. Ampicillin, kanamycin 등에 resistance gene 3) Multiple cloning site(MCS) 제한효소로 잘릴 수 있는 부위를 한꺼번에 모아놓은 부분. 몇 가지 제한효소 절단부위를 인위적으로 조작 해 놓은 부분.

7 Competent cell (CP cell)
Competence is the ability of cells to take up exogenous DNA from the environment Two types of competence: Natural competence : Bacteria have cellular machinery to take up DNA from environment Artificial competence : Cells are made competent in the laboratory allowing them to take up DNA

8 ▶▷ Competent cell Competent cell (CP cell) 이란?
- 정상적인 bacteria에 화학적 처리를 하여 DNA가 잘 들어갈 수 있게 만든 cell. - 보통 사용하는 E.coli 균주는 M13 phage DNA 경우 XL1-Blue가 사용되며, plasmid transformation을 위한 균주는 DH5a 또는 XLA-Blue 등이 사용된다. Competent cell에 화학처리에 쓰이는 시약의 종류 ( divalent cation등에 의해 세포막 고유 charge가 변하게 됨에 따라, 막구조의 불안정이 야기되어 흐물흐물한 상황이 됨.) - Calcium Chloride    - Manganase Chloride    - Hexamminecobalt Chloride    - Dimethyl Sulfoxide (DMSO) 만들어진 Competent cell은 -70℃에 보관하고 필요할 때 꺼내서 ice에 있는 상태에서 녹인 후 Transformation에 쓰게 된다.

9 Competent cell (CP cell)
1) negative charge를 띠는 DNA와 마찬가지로 negative charge를 가지는 E. coli cell surface는 서로 척력을 가져서 DNA가 cell 안으로 들어가기 힘들어진다. 2) CaCl2 처리해 Competent cell을 만들 때에는 Ca2+ ion으로 cell surface를 coating하여 cell surface에 DNA가 잘 붙게 한다. 3) Heat shock은 DNA를 활발하게 움직이게 해서 cell 안으로 잘 들어가게 하는 역할과 cell 표면의 DNA가 들어갈 수 있는 구멍을 넓히는 역할을 해서 Transformation의 efficiency를 높인다.

10 Selection Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or have their growth arrested.  Antibiotic resistance is the most commonly used marker for prokaryotes.

11 Transformation

12 Transfection

13 Materials - DNA ( with Ampicillin Resistance )
- Competent Cell ( incubation in ice )   - Water Bath ( preset to 42℃ ) - Spreader ( in 100% Ethanol )   - Alcohol Lamp   - LB Plate ( Ampicillin Resistance ) LB 배지 + Amp (100 ug/ml) LB 배지 Trypton(10g/L): amino acids와 peptides 공급 Yeast Extract(5g/L); 질소, 당, 무기, 유기물 등을 제공 NaCl(10g/L); 세포 자체 내에서 ion을 사용하여 신호전달단계에서 필요 Agar(5g/L); 고체 배지를 만들 때 배지를 굳힘. 액체 배지를 만들 때는 넣지 않음.

14 Method 준비된 competent cell을 얼음에서 녹인다.
Competent cell에 DNA를 2ml 넣고 tip으로 저어준다. CP cell을 ice에서 30분(대략 15분이상) 동안 incubation Heat block에서 42℃ 1분heat shock Ice에서 20분(대략 10분이상)간 incubation 액체 LB negative 배지 900 ul를 넣고 37 ℃, shaking incubator에서 30-60분간 regeneration 7. Spin down 후 soup 버리기 8. 남은 LB로 pellet을 pipetting 9. LB+항생제 배지(AmpR)에 spreading