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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
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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
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Restriction Analysis of pDRK and pGRN Restriction Fragments after digest with HindIII
kanr ara C PBAD ampr gfp
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Restriction Analysis of pDRK and pGRN Prediction for restriction gel
M D+ D- G+ G-
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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
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Ligation of pDRK/pGRN Restriction Fragments Recombinant Plasmid of Interest
PBAD ampr ara C HindIII gfp
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Restriction Analysis of pDRK and pGRN Confirmation of restriction and ligation
M D+ D- G+ G- L
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Preparing Competent Cells for Transformation
++ - Lipid bilayer inner membrane outer membrane Peptidoglycan layer adhesion zone phospholipids calcium ion
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Transforming Escherichia coli With a Recombinant Plasmid
Recombinant plasmids transformed cells Competent cells
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Transforming Escherichia coli With a Recombinant Plasmid
++ adhesion zone ++ ++ ++ - ++ ++ Lipid bilayer inner membrane ++ ++ Peptidoglycan layer ++ ++ Lipid bilayer outer membrane ++ ++ phospholipids ++ ++ ++ ++ ++ ++ calcium ion ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ plasmid
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Growth of transformed bacteria on various plates
P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp
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Preparing an Overnight Culture of E
Preparing an Overnight Culture of E. coli for GFP Expression Colony isolation and culture
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GFP Expression ara C gene gfp gene promoter transcription mRNA
translation C protein
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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
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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
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GFP
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Purification of GFP from an Overnight Culture
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