Restriction Analysis of pDRK and pGRN Original Plasmids Hin dIII Hin dIII pGRN Hin dIII pDRK ara C amp r kan r gfp P BAD.

Restriction Analysis of pDRK and pGRNOriginal Plasmids

HindIII

HindIII

pGRN

HindIII

pDRK

ara C

ampr

kanr

gfpPBAD

Restriction Analysis of pDRK and pGRNRestriction Fragments after digest with HindIII

gfp

ara C

PBAD ampr

kanr

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

Preparing Competent Cells for Transformation

++

---

--

-- -

----

---

Lipid bilayerinner membrane

Lipid bilayerouter membrane

Peptidoglycan layer

++++

++

++

++++

++

++

++++ ++ ++

++

++++

adhesion zone

phospholipids

calcium ion

++

---

--

--- -

----

---

Lipid bilayerinner membrane

Lipid bilayerouter membrane

Peptidoglycan layer

++

++

++++

++

++

++++

++

++

++++ ++ ++

++

++

++++

++

++

++

++++

++

++

++

++++

plasmid

adhesion zone

phospholipids

calcium ion

Transforming Escherichia coli With a Recombinant Plasmid

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

Transforming Escherichia coli With a Recombinant Plasmid

Recombinant plasmids

Competent cells

transformed cells

Growth of transformed bacteria on various plates

P+ plates

P- plates

LB LB/amp LB/amp/ara

LB LB/amp

No growth

GFP

Ligation of pDRK/pGRN Restriction FragmentsRecombinant Plasmid of Interest

rpGLO PBAD

ampr

ara C

HindIII

HindIII

gfp