Restriction Analysis of pDRK and pGRN Original Plasmids ara C H i n d I I I ampr PBAD gfp p D R K p G R N H i n d I I I H i n d I I I kanr

Restriction Analysis of pDRK and pGRN Restriction Fragments after digest with HindIII kanr ara C PBAD ampr gfp

Ligation of pDRK/pGRN Restriction Fragments DNA ligase A A G C T T T T C G A A G C T T C G A gfp

Ligation of pDRK/pGRN Restriction Fragments Recombinant Plasmid of Interest PBAD ampr ara C HindIII gfp

Preparing Competent Cells for Transformation ++ - Lipid bilayer inner membrane outer membrane Peptidoglycan layer adhesion zone phospholipids calcium ion

Transforming Escherichia coli With a Recombinant Plasmid ++ adhesion zone ++ ++ ++ - ++ ++ Lipid bilayer inner membrane ++ ++ Peptidoglycan layer ++ ++ Lipid bilayer outer membrane ++ ++ phospholipids ++ ++ ++ ++ ++ ++ calcium ion ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ plasmid

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 C protein RNA polymerase 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 transformed cells Competent cells

Growth of transformed bacteria on various plates P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp

GFP

Ligation of pDRK/pGRN Restriction Fragments Recombinant Plasmid of Interest PBAD ampr ara C HindIII gfp