复旦大学：《医学微生物学 Medical Microbiology（MBBS）》课程讲义_实验课_Plasmid Transformation

Plasmid transformation Zhang Junqi, Han Wendong 2012-10-11

1 Plasmid Transformation Zhang Junqi, Han Wendong 2012-10-11

1. Experiments: Transformation of plasmid dna 2. Demonstration Plasmid extraction

2 1. Experiments: Transformation of Plasmid DNA 2. Demonstration: Plasmid extraction

Characteristics of plasmids Plasmid is a type of dna existence within bacteria >extrachromosomal genomes >self-replicating >commonly used vectors

3 Characteristics of plasmids Plasmid is a type of DNA existence within bacteria extrachromosomal genomes self-replicating commonly used vectors

Some bacterial characteristics are determined by plasmids L Antibiotic resistance Many bacteria can become resistant to antibiotics by acquisition of a plasmid 2. Colicins and bacteriocins Another property conferred by some plasmids that has been widely studied is the ability to produce a protein which has an antimicrobial action 3. Virulence determinants In some bacterial species toxin genes are carried on plasmids

4 Some bacterial characteristics are determined by plasmids 1.Antibiotic resistance Many bacteria can become resistant to antibiotics by acquisition of a plasmid 2.Colicins and bacteriocins Another property conferred by some plasmids that has been widely studied is the ability to produce a protein which has an antimicrobial action 3.Virulence determinants In some bacterial species toxin genes are carried on plasmids

Nucleoid Plasmids Plasmids are small circular DNA molecules that are found inside some prokaryotic cells schematic diagram of plasmid

5 schematic diagram of plasmid

ransformation Gene Transfer (I)Transformation, in which a cell takes up isolated DNA molecules from the medium surrounding it (2) Conjugation () Transduction

6 Transformation Gene Transfer (1)Transformation, in which a cell takes up isolated DNA molecules from the medium surrounding it. (2) Conjugation (3) Transduction

The developing prosess of transformation 1928, Fred griffith, working with pneumococcus(肺炎链球菌) avirulent strains could be restored to virulence by incubation with an extract from killed virulent cells 1944, Avery, macleod and mc carty demonstrated that the transforming principle waS DNA 1970, Mandel and hlga did the transformation by competent cells (chemical method, CaCl, deal with cells Now, transformation is very important because of its key role in gene cloning

7 The developing prosess of transformation 1928，Fred Griffith, working with pneumococcus(肺炎链球菌) avirulent strains could be restored to virulence by incubation with an extract from killed virulent cells. 1944，Avery, MacLeod and McCarty demonstrated that the ‘transforming principle’ was DNA. 1970，Mandel and Hlga did the transformation by competent cells （chemical method，CaCl，deal with cells ） Now， transformation is very important because of its key role in gene cloning

<dp Centrifuge Resuspend in Log phase culture nd in small volume Plasmid DNA Dilute and plate on Add broth selective medium Incubate to allow gene Heat ession shock Antibiotic-resistant 8

8 How to get competent cells？ transformation

Experiment Transformation of plasmids into e coli Purpose To utilize competent E. coli bacteria to replicate a specific DNA fragment

9 Experiment: Transformation of Plasmids into E. coli Purpose: To utilize competent E. coli bacteria to replicate a specific DNA fragment

Anti amp plasmid competent E coli LB/Amp plates

10 Anti Amp plasmid competent E. coli LB/Amp plates