Transformation Biology experiment Historical Perspective Frederic Griffith 1928

Transformation Biology experiment

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

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 Prokaryote Eukaryote DNA Term Plasmid Transformation Bacteriophage (virus) Infection Plasmid Transfection Virus vector Transfection

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

Plasmid Small circular ds. DNA 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 (<10 kb insertion) • •

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

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.

Transformation

Transfection

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(10 g/L): amino acids와 peptides 공급 Yeast Extract(5 g/L); 질소, 당, 무기, 유기물 등을 제공 Na. Cl(10 g/L); 세포 자체 내에서 ion을 사용하여 신호전달단계에서 필요 Agar(5 g/L); 고체 배지를 만들 때 배지를 굳힘. 액체 배지를 만들 때는 넣지 않음.

Method 1. 준비된 competent cell을 얼음에서 녹인다. 2. Competent cell에 DNA를 2 ml 넣고 tip으로 저어준다. 3. CP cell을 ice에서 30분(대략 15분이상) 동안 incubation 4. Heat block에서 42℃ 1분heat shock 5. Ice에서 20분(대략 10분이상)간 incubation 6. 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(Amp. R)에 spreading