武汉大学生命科学学院：《分子生物学》英文版 Gene cloning, expression and functional study 基因克隆，表达及功能研究

Gene cloning, expression and functional stud 基因克隆,表达及功能研究

Gene cloning, expression and functional study 基因克隆，表达及功能研究

vectors Cloning vectors:克隆载体 to clone a gene in a vector Expression vectors:表达载体 to express a gene from a vector Integration vectors:整合载体 to integrate a gene in a genome through a vector

vectors Cloning vectors: 克隆载体 to clone a gene in a vector Expression vectors: 表达载体 to express a gene from a vector Integration vectors: 整合载体 to integrate a gene in a genome through a vector

Cloning vectors 1 Plasmid vecters 2 Bacteriophage vectors 3 Cosmids BACs 4 Eukaryotic vectors

Cloning vectors 1 Plasmid vecters 2 Bacteriophage vectors 3 Cosmids & BACs 4 Eukaryotic vectors

Cloning vectors: allowing the exogenous dna to be inserted, stored and manipulated mainly at DNa level expression vectors: allowing the exogenous dna to be inserted, stored and expressed

Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. expression vectors: allowing the exogenous DNA to be inserted, stored, and expressed

A plasmid vector for cloning 1. Contains an origin of replication, allowing for replication independent of host's genome. 2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3. Contains a multiple cloning site(MCS 4. easy to be isolated from the host cell

1. Contains an origin of replication, allowing for replication independent of host’s genome. 2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistance blue-white screening 3. Contains a multiple cloning site (MCS) 4. Easy to be isolated from the host cell. A plasmid vector for cloning

Twin antibiotic resistance screening Screening by insertional inactivation of a resistance gene Amp B X B pBR322 B Amp T Ampr or Ampicillin resistant? es ye yes Tetracycline resistant No yes

Ampicillin resistant? yes yes Tetracycline resistant? No yes B X B B B X Ampr ori Ampr Tcr ori pBR322 Ampr Tcr ori -Screening by insertional inactivation of a resistance gene Twin antibiotic resistance screening

Replica plating: transfer of the colonies from one plate to another using absorbent pad or velvet($1p) transfer of colonies +ampicillin ampicillin tetracycline these colonies have bacteria with recombinant plasmid

Replica plating: transfer of the colonies from one plate to another using absorbent pad or Velvet (绒布). transfer of colonies +ampicillin + ampicillin + tetracycline these colonies have bacteria with recombinant plasmid

Blue white screening Screening by insertional inactivation of the lacz gene Lac promoter Ampr Mcs(Multiple cloning sites 多克隆位点) pUC18 lacz (3kb) orl The insertion of a dna fragment interrupts the ORF of lacz" gene, resulting in non-functional gene product that can not digest its substrate x-gal

Blue white screening Ampr ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位点） Lac promoter lacZ’ Screening by insertional inactivation of the lacZ gene The insertion of a DNA fragment interrupts the ORF of lacZ’ gene, resulting in non-functional gene product that can not digest its substrate x-gal

Recreated vector: blue transformants Recombinant plasmid containing inserted dNa white transformants Recreated vector(no insert) Recombinant plasmid (contain insert back

Recreated vector: blue transformants Recombinant plasmid containing inserted DNA: white transformants Recreated vector (no insert) Recombinant plasmid (contain insert) back

Multiple cloning sites Multiple restriction sites enable the convenient insertion of target DNA into a vector Lac promoter Amp MCS(Multiple cloning sites 多克隆位点) pUC18 lacz (3kb) or Sall Smal Hindi EcorI SacI KpnI Xmal BamhI XbaI Accl PstI SphI ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA ThrAsn ser s e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser Lac z

Multiple cloning sites Multiple restriction sites enable the convenient insertion of target DNA into a vector Ampr ori pUC18 (3 kb) MCS (Multiple cloning sites, 多克隆位点） Lac promoter lacZ’ …ACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA… . T h rA s n S er S e r Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser… EcoRI SacI KpnI SmaI XmaI BamHI XbaI SalI HincII AccI PstI SphI Lac Z