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Download COMBINING GENES FROM TWO PLASMIDS
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Restriction Analysis of pDRK and pGRN Original Plasmids ara C HindIII ampr gfp pGRN HindIII kanr pDRK PBAD HindIII Restriction Analysis of pDRK and pGRN Restriction Fragments after digest with HindIII kanr ara C PBAD gfp ampr Ligation of pDRK/pGRN Restriction Fragments DNA ligase TCGAA AGCTT gfp TTCGA A AGCT DNA ligase Ligation of pDRK/pGRN Restriction Fragments Recombinant Plasmid of Interest ara C ampr PBAD rpGLO HindIII gfp HindIII Preparing Competent Cells for Transformation ++ - - - - - - -++ calcium ion ++ adhesion zone - - ++ - - ++ ++ ++ ++ - ++ - ++ ++ Lipid bilayer inner membrane Peptidoglycan layer ++ Lipid bilayer outer membrane ++ ++ ++ ++ phospholipids Transforming Escherichia coli With a Recombinant Plasmid ++ - - - - - - -++ calcium ion ++ adhesion zone - - ++ - ++ ++ ++ ++ - ++ - ++ Lipid bilayer inner membrane Peptidoglycan layer ++ Lipid bilayer outer membrane ++ - - ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ plasmid ++ phospholipids GFP Expression ara C gene gfp gene promoter transcription mRNA translation C protein GFP Expression ara C gene gfp gene promoter C protein prevents gfp transcription by causing a loop to form in the region of the gfp gene GFP Expression arabinose RNA polymerase C protein arabinose – C protein complex ara C gene gfp gene promoter transcription mRNA Arabinose – C protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site. translation GFP Transforming Escherichia coli With a Recombinant Plasmid Recombinant plasmids Competent cells transformed cells Growth of transformed bacteria on various plates P+ plates LB P- plates LB/amp No growth LB LB/amp LB/amp/ara GFP Ligation of pDRK/pGRN Restriction Fragments Recombinant Plasmid of Interest ara C ampr PBAD rpGLO HindIII gfp HindIII
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