pARA-R RecombinantRFP Expression Sequence

biotechnology lab program

Laboratory Protocols by:

Marty IkkandaPowerpoint by:

Anthony DauloPierce College, Woodland Hills, CA

V.1.2.1

restriction analysis of pKAN-R and pARA

pKAN-R

pARA

BamH I

Hind III

BamH IHind III

rfp702 bp

5408 bp

4058 bp

40 bp

restriction analysis of pKAN-R and pARA

BamH I Hind III

702bp

40 bp

BamH I Hind III

BamH IHind III

BamH IHind III

Restriction fragments after digest with Hind III and BamH I

4018 bp

4706 bp

restriction analysis of pKAN-R and pARA

Prediction for restriction gel

M K+ K- A+ A-M K+ K- A+ A-

500

1000

1500

20003000400050008000

10000

ligation of pKAN-R/pARA restriction fragments

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

ligation of pKAN-R/pARA restriction fragments

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

restriction analysis of pKAN-R and pARA

M K+ K- A+ A-

500

1000

1500

20003000400050008000

10000

Confirmation of restriction and ligation

L M K+ K- A+ A- L

preparing competent cells for transformation

Lipid bilayer(inner)

Lipid bilayer(outer)

Peptidoglycanlayer

Adhesion zone

Calcium ions

transforming Escherichia coli with pARA-R

Recombinant Plasmids

Competent Cells

pARA-R

Calcium ions

pARA-R

transforming Escherichia coli with pARA-R

Adhesion zone

Lipid bilayer(inner)

Lipid bilayer(outer)

Peptidoglycanlayer

growth of transformed bacteria on various plates

P+ plates

P- plates

LB LB/amp LB/amp/ara

LB LB/amp

No growth

Colony isolation and culture

preparing an overnight culture of E. Coli for RFP expression

LB/amp/arabroth

RFP expression

araC gene rfp genePBAD

Transcription

mRNA

Translation

araC protein

RFP expression

rfp genePBAD

araC protein

araC gene

araC protein prevents RFP transcription by causinga loop to form in the region of the fp gener

RFP expression

araC protein

arabinose

araC gene rfp genePBAD

arabinose – araC proteincomplex

RNA polymerase

Arabinose – araC protein complex prevents DNA loopingand helps to align RNA polymerase

on the promoter site (PBAD).

mRNA

Transcription

Translation

RFP(red fluorescent

protein)

RFP

purification of RFP from an overnight culture

Overnightculture

Cell pelletwith RFP

Lysedcells

Pelletcell debris

RFP withbinding buffer