北京大学：《微生物学》课程PPT教学课件（英文版）Genetic Engineering and Biotechnology

Genetic Engineering and Biotechnology Genetic engineering: Isolation, manipulation and expression of genetic materials Gene cloning: Isolation and purification of specilic genes In vitro recombination=recombinant dNa

Genetic Engineering and Biotechnology Genetic engineering: Isolation, manipulation and expression of genetic materials. Gene cloning: Isolation and purification of specific genes In vitro recombination=recombinant DNA

Gene Cloning Procedures Isolation and fragmentation of the source DNA Joining the dna fragments to a cloning vector with DNA ligase Incorporation into a host Detection and purification of the desired clone Production of large numbers of cells or bacteriophage containing the desired clone for isolation and study of the clone dna

Gene Cloning Procedures Isolation and fragmentation of the source DNA Joining the DNA fragments to a cloning vector with DNA ligase Incorporation into a host Detection and purification of the desired clone Production of large numbers of cells or bacteriophage containing the desired clone for isolation and study of the clone DNA

Plasmids as Cloning Vectors Clal Small, only 4361 BP. EcoRI Hind Ill Stable in E coli BamHI High copy number(1000-3000 copies per cell) Easily isolated in the Ampicillin Sa/I esistance supercoiled form Plasmid pBR322 Foreign DNA can be inserted in good amount Restriction sites are known Single cleavage sites for several Tetracycline resistance restriction enzymes Two antibiotic resistance Origin of DNA replication markers Transformation easy

Plasmids as Cloning Vectors Small, only 4361 BP, Stable in E. coli, High copy number (1000-3000 copies per cell) Easily isolated in the supercoiled form Foreign DNA can be inserted in good amount Restriction sites are known Single cleavage sites for several restriction enzymes Two antibiotic resistance markers Transformation easy Plasmid pBR322

BamHI site Gene cloning and Expression Tc pBR322 using Plasmid pBr322 BamHI sites BamHI digestion Foreign DNA BamHI digestion DNA ligase Recyclized Hybrid containing foreign DNA pBR322 Transformation of E coli Transformants Transformants resistant to both resistant to ampicillin ampicillin and tetracycline but sensitive to tetracycline

Gene cloning and Expression using Plasmid pBR322

Bacteriophages as Cloning Vectors Modified lambda hages as cloning vectors pl 80 100 Capsid components J b region att int xis N P c POP OSR anoo Replaceable region Charon 4A B-Gal gene Another substitution Charon 16

Bacteriophages as Cloning Vectors Modified lambda phages as cloning vectors

Nonessential region Cosmids: plasmid vectors containing foreign DNA Digestion w ends restrictio plus only the cos(cohesive end) site from the lambda genome Hynd DNA Sizes of a cloning gene the Packaging with vectors can carry YAC>Cosmid>Lambda> Plasmids

Cosmids: plasmid vectors containing foreign DNA plus only the cos (cohesive end) site from the lambda genome Sizes of a cloning gene the vectors can carry: YAC>Cosmid>Lambda>Plasmids

Other vectors Yeast artificial chromosomes (YAC) TEL ARS CEN Not I Not I URA3 INSERTED DNA YAC vectors are only about 10 KB, but can carry 200-800 KB DNA sequences YAC has been developed and used for the human genome project

Other Vectors Yeast artificial chromosomes (YAC) YAC vectors are only about 10 KB, but can carry 200-800 KB DNA sequences YAC has been developed and used for the human genome project

Vectors for DNA Sequencing: Bacteriophage M13 Phagemid: a hybrids ICG AGC TCG GTA CCC GGG GAT CCT CTA GAG TCG ACC TGC AGG CAT GCA A between a Asn SerSerSer Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser filamentous phage, like Mi3 and a plasmid Vectors with cloned DNA Vectors without cloned dNA X-gal: 5-bromo-4 hloro-3-indolyl-B-D galactopyranoside

Vectors for DNA Sequencing: Bacteriophage M13 Phagemid: a hybrids between a filamentous phage, like M13 and a plasmid X-gal: 5-bromo-4- chloro-3-indolyl-b-D￾galactopyranoside Vectors without cloned DNA Vectors with cloned DNA

Other specialized vectors Expression vectors: to obtain synthesis of the protein coded for by the foreign gene cloned into the vector Secretion vectors: protein product is not only expressed but secreted (excreted) from the cell Shuttle vectors: move dna between unrelated organisms, can replicate in two different organisms

Other specialized vectors Expression vectors: to obtain synthesis of the protein coded for by the foreign gene cloned into the vector. Secretion vectors: protein product is not only expressed but secreted (excreted) from the cell. Shuttle vectors: move DNA between unrelated organisms, can replicate in two different organisms

Hosts for cloning vectors Ideal hosts: rapid growth, capable of growth in cheap culture medium, not harmful or pathogenic transformable by dNa, stable Prokaryotic hosts: E coli, Bacillus subtilis Eukaryotic hosts: Saccharomyces cerevisiae DNA vIrus sv40. a virus causing tumors in primates, can be used as a cloning vector into human culture lines Retroviruses. vaccinia virus are useful too Baculovirus. an insect DNA virus can be used to transfer dna to insect cell lines

Hosts for cloning vectors Ideal hosts: rapid growth, capable of growth in cheap culture medium, not harmful or pathogenic, transformable by DNA, stable. Prokaryotic hosts: E. coli, Bacillus subtilis. Eukaryotic hosts: Saccharomyces cerevisiae. DNA virus SV40, a virus causing tumors in primates, can be used as a cloning vector into human culture lines. Retroviruses, vaccinia virus are useful too. Baculovirus, an insect DNA virus can be used to transfer DNA to insect cell lines