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