Pierce College, Woodland Hills Bruce Wallace Biotechnology Laboratory Program rpGLO Recombinant – GFP Expression Sequence                                                                              Laboratory protocols by: Marty Ikkanda PowerPoint by: Lyn Koller Pierce College, Woodland Hills

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

Restriction Analysis of pDRK and pGRN Prediction for restriction gel M D+ D- G+ G-

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

Restriction Analysis of pDRK and pGRN Confirmation of restriction and ligation M D+ D- G+ G- L

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 Recombinant plasmids transformed cells Competent cells

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

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

Preparing an Overnight Culture of E Preparing an Overnight Culture of E. coli for GFP Expression Colony isolation and culture

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

GFP

Purification of GFP from an Overnight Culture