Bruce Wallace Biotechnology

Laboratory Program

rpGLO Recombinant – GFP Expression Sequence

PowerPoint by:

Lyn Koller

                                             

Laboratory protocols by:

Marty Ikkanda

Pierce College, Woodland Hills

                              

Restriction Analysis of pDRK and pGRN

Original Plasmids

HindIII

HindIII

pGRN

HindIII

pDRK

ara C

ampr

kanr

gfpPBAD

Restriction Analysis of pDRK and pGRN

Restriction Fragments after digest with HindIII

gfp

ara C

PBAD ampr

kanr

Restriction Analysis of pDRK and pGRN

Prediction for restriction gel

M D+ D- G+ G-

Ligation of pDRK/pGRN Restriction Fragments

DNA ligase

AA G C T T A

T T C G A A G C T

T C G A

DNA ligase

gfp

Ligation of pDRK/pGRN Restriction FragmentsRecombinant Plasmid of Interest

rpGLO PBAD

ampr

ara C

HindIII

HindIII

gfp

Restriction Analysis of pDRK and pGRN

Confirmation of restriction and ligation

M D+ D- G+ G- L

Preparing Competent Cells for Transformation

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Lipid bilayerinner membrane

Lipid bilayerouter membrane

Peptidoglycan layer

++++

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++++ ++ ++

++

++++

adhesion zone

phospholipids

calcium ion

Transforming Escherichia coli With a Recombinant

Plasmid

Recombinant plasmids

Competent cells

transformed cells

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Lipid bilayerinner membrane

Lipid bilayerouter membrane

Peptidoglycan layer

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++

++++

++

++

++++

++

++

++++ ++ ++

++

++

++++

++

++

++

++++

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++

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++++

plasmid

adhesion zone

phospholipids

calcium ion

Transforming Escherichia coli With a Recombinant

Plasmid

Growth of transformed bacteria on various plates

P+ plates

P- plates

LB LB/amp LB/amp/ara

LB LB/amp

No growth

Preparing an Overnight Culture of E. coli for GFP

ExpressionColony isolation and culture

GFP Expression

ara C gene

gfp gene

promoter

mRNA

transcription

translation

C protein

GFP Expression

C protein prevents gfp transcription by causing a loop to form in the region of the gfp gene

ara C gene

gfp gene

promoter

GFP Expression

ara C gene gfp gene

promoter

arabinose

mRNA

Arabinose – C protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site.

transcription

translation

GFP

RNA polymerase

arabinose – C protein complex

C protein

GFP

Purification of GFP from an Overnight Culture