1. 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

2. 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

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

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

5. 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

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

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

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

9. Transforming Escherichia coli With a Recombinant Plasmid
Recombinant plasmids
transformed cells
Competent cells

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

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

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

13. GFP Expression ara C gene gfp gene promoter transcription mRNA
translation
C protein

14. 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

15. 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

16. GFP

17. Purification of GFP from an Overnight Culture