1. pARA-R Sequence LABS 2a and 4a RFP Expression Sequence
biotechnology lab program
LABS 2a and 4a
pARA-R Sequence
RFP Expression Sequence
Laboratory Protocols by:
Marty Ikkanda
Powerpoint by:
Anthony Daulo
Kristi Schramm
Pierce College, Woodland Hills, CA
V.1.2.1

2. Engineering the Plasmid: ligation of rfp gene into p-ARA
sticky end
BamH I
sticky end
Hind III
sticky end
Hind III
sticky end
BamH I

3. Recombinant plasmid of interest
Restriction digest of pARA-R
Sites labeled BamH I and Hind III represent restriction sites for these restriction enzymes
Recombinant plasmid of interest
pARA - R contains a gene for ampicillin
resistance, ampr
pARA-R
4720 bp
rfp
702bp
BamH I
Hind III

4. Red sea anemone aids biomedical research
Red sea anemone aids biomedical research. A new red fluorescent protein derived from a bright red sea anemone discovered in the tropical ocean.

5. The fluorescent protein gene was originally isolated from Discosoma , a sea anemone, which is a Eukaryotic organism.
Therefore, the recombinant DNA plasmid, pARA-R has elements of both Prokaryotic and Eukaryotic Organisms.
The pARA-R has been engineered into the bacterial plasmid to express the rfp gene to produce a mutant RED FLUORESCENT PROTEIN (mFP); known as GENE EXPRESSION

6. Restriction fragments after digest with Hind III and BamH I
restriction analysis of pARA-R
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
Hind III
BamH I
702bp

7. Prediction for restriction gel
restriction analysis of pARA-R
Prediction for restriction gel M A+ A- M A+ A-
500
1000
1500
2000
3000
4000
5000
8000
10000
4.018kb
0.702kb

8. Lab 5a - Recombinant plasmid of interest
pARA-R construct
Lab 5a - Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp
702bp
Hind III

9. LAB 5A – Transformation of Escherichia coli (E. coli)
A REVIEW 
Recombinant DNA Plasmid
Bacterial transformation involves transfer of genetic information into a cell by direct uptake of the DNA.
Transformation can occur naturally but the incidence is very low. These bacterium can take up DNA only during
the period of logarithmic growth. At this time the cells are said to be competent. Once competent, the cells are ready to accept DNA. (see clip)
Restriction enzymes (Endonucleases) can be used to cut and insert pieces of foreign DNA into the plasmid vector - a self replicating DNA molecule.
** Gene Cloning Using Plasmids - reading assignment

10. preparing competent cells for transformation
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions

11. transforming Escherichia coli with pARA-R
Competent Cells
pARA-R
Recombinant Plasmids

12. transforming Escherichia coli with pARA-R
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
pARA-R

13. Factors that determine transformation
The larger the plasmid, the less likely it will be taken up by the bacterium. supercoiled form is the easiest.
Shape of the plasmid - supercoiled, nicked circle, multimer. Supercoiled form is the easiest to pass through the membrane.
Making Competent Cells
Suspend (soak) in calcium chloride to neutralize the negative electrical charges of the plasma membrane.
Create pressure difference between the inside and outside of the bacterial cell - “heat shock”, really hot and then really cold.

14. The bacterial plasmid
ampr - ampicillin resistant gene - prevents bacteria from fully forming their cell walls.
Arabinose - a simple sugar, is needed by the bacterium to express the rfp gene.
PBAD - where the RNA polymerase needs to bind to initiate transcription

15. Answer the following questions before beginning the lab.
What is transformation?
What is a competent cell and how are competent cells produced?
What is heat shock?
What is a recombinant plasmid?
What is the function of Ampicillin?
What is the function of arabinose?
Making Competent Cells 1.

16. Preparing Competent Cells for Transformation
(re-suspended cells)
pARA-R
(plasmid)
*AFTER 15 minutes in ice
LB broth
“LB” P+ 50
Microliters
0-5-0
(P-200)
10 Microliters
1-0-0
(P-20)
150
1-5-0 P-
Negative control
50 Microliters
150 Microliters

17. PLATE PREPARATION P- P+ P- P+ P+ LB
LB/amp
LB/amp/ara
Draw a line on the bottom of the LB and LB/amp plates - PREPARE PLATES DURING ZERO PERIOD

18. Results P+
P- P+
P P+ _ + + + + LB
LB/amp
LB/amp/ara

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

20. LAB 6 - Colony isolation and culture
preparing an overnight culture of E. Coli for RFP expression
LAB 6 - Colony isolation and culture
LB/amp/ara
broth

21. RFP expression araC protein prevents RFP transcription by causing
a loop to form in the region of the fp gene r
araC gene
araC protein
PBAD
rfp gene

22. RFP expression araC gene PBAD rfp gene Transcription mRNA Translation
araC protein

23. arabinose – araC protein
RFP expression
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
rfp gene
PBAD

24. RFP

25. RFP

26. purification of RFP from an overnight culture
Cell pellet
with RFP
Lysed
cells
Pellet
cell debris
RFP with
binding buffer