Restriction Analysis of pDRK and pGRN Original Plasmids

1. Restriction Analysis of pDRK and pGRNOriginal 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

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

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

4. ara C ampr r p G L O PBAD HindIII gfp HindIII Ligation of pDRK/pGRN Restriction FragmentsRecombinant Plasmid of Interest

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

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

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

8. 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

9. arabinose RNA polymerase C protein arabinose – C protein complex transcription mRNA translation ara C gene gfp gene GFP promoter GFP Expression Arabinose – C protein complex prevents DNA looping and helps to align RNA polymerase on the promoter site.

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

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

12. GFP

13. ara C ampr r p G L O PBAD HindIII gfp HindIII Ligation of pDRK/pGRN Restriction FragmentsRecombinant Plasmid of Interest