1. Abridged Genetic Engineering Pathway (Original “A” Sequence)

2. Recombinant Plasmid pARA-R

3. Restriction fragments after digest with HindIII and BamHI
Restriction Analysis of pARA-R
Restriction fragments after digest with HindIII and BamHI
BamHI
HindIII
araC
ori
ampR
4,495 bp
HindIII
BamHI
pBAD
807 bp

4. Confirmation of Restriction
Restriction analysis of pARA-R (cont.)
Bruce Wallace
Confirmation of Restriction
Prediction for restriction gel
Results for restriction gel M R+ R– M R+ R–
500
1000
1500
2000
3000
4000
5000
8000
10000

5. Preparation of Competent Cells
Add calcium chloride
Heat shock

6. Transformation of E. coli with pARA-R
Competent Cells
pARA-R
Recombinant Plasmids

7. Transformation of E. coli with pARA-R
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
pARA-R

8. Growth and Selection of Transformed Bacteria
P+ plates LB
LB/amp
LB/amp/ara
P– plates
No growth LB
LB/amp

9. Overnight Culture of E. coli for rfp Expression
Colony isolation and culture
LB/amp/ara
broth

10. arabinose – araC protein
RFP Production
RFP
(red fluorescent
protein)
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
arabinose
Translation
RNA polymerase
arabinose – araC protein
complex
araC protein
mRNA
Transcription
araC gene
PBAD
rfp gene

11. RFP Purification Overnight culture Cell pellet with RFP Lysed cells
cell debris
RFP with
binding buffer

12. Three-dimensional Shape of RFP