1. Lab 5a Transformation of Escherichia coli with pARA-R

2. The “acid test” – does it work?
“transformed cell” – cell has acquired new characteristics
“characteristics” – due to the expression of incorporated foreign genetic material
The “acid test” – does it work?

3. The Big Picture
2005 Pearson Education, Inc.

4. Differential gene expression
Gene expression – process by which the information encoded in a gene is converted into an observable phenotype
Gene regulation – control mechanisms that turn genes on or off
Inducible proteins – synthesis is regulated depending on the bacterium’s nutritional status
Thank you Francois Jacob and Jacques Monod!
Prokaryote operon model of gene control
Repressors and activators are “trans-acting” – affect expression of their genes no matter on which DNA molecule in the cell these are located.

5. Why do we need arabinose?
Why don’t we see the red protein in any LB growth media?
Cells conserve energy and resources
The rfp gene requires a specific substrate (arabinose) to be turned on (expressed)

6. Recombinant plasmid of interest
pARA-R construct
Recombinant plasmid of interest
pARA-R
4720 bp
BamH I
rfp
702bp
Hind III

7. RFP expression araC gene PBAD rfp gene Transcription mRNA Translation
Rfp gene is in place of the araA, araB and araD genes which produce enzymes that digest arabinose in the normal arabinose operon.
Translation
araC protein

8. 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

9. RFP expression
Rfp gene has replaced the araA, araB, and araD genes in the normal arabinose operon. These produce enzymes that digest arabinose.
araC gene
araC protein
PBAD
araB
araA
araD
rfp gene

10. 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
Arabinose is the inducer
araC gene
rfp gene
PBAD

11. What do we know?
1. Plasmid with gene of interest has been produced – confirmed by gel electrophoresis
What do we want to know?
2. Can the plasmid (vector) be taken in by a host cell (E. coli)?
3. Can the host cell express the gene of interest and produce a product that can be utilized?

12. How is Lab 5a different from Lab 5?
“Materials” changes for “a” strand folks: Instead of ‘E. Coli plate’ you will use: 100 uL of competent cells Instead of “1 mL 50 mM CaCl2” you will use: 350 uL of LB broth (sterile)

13. Agar Plate tips to tell the students:
Note the plate markings: I=LB, II=LB/amp, III=LB/amp/ara
Label the bottom of the plate near the edge
Open the plates like clam shells
Sample goes on the agar, not the lid
(really, you need to remind them)

14. Agar is like finger jello, firm but not invincible, be gentle – the “spreader is not a shovel
Turn the plates upside down (lids down) for incubation, stacked and taped together
After incubation, do not open plates, observe through the bottom

15. Three Important aspects to stress with your students
1. Sterile technique
Using bacteria
Contamination may affect results
2. Carefully READ and FOLLOW the lab protocol.
Be sure lab partners communicate
3. No Food or Drinks

16. Sterile technique tips for students
Always follow the protocol carefully – know what you’re doing
Work quickly – less time = less opportunities for contamination
Do not leave any container (tube, plate) open any longer than needed
Watch what your equipment touches – there is no “5 second rule” here.
All tips, tubes and spreaders go in the “contaminated waste” container at the end of the lab.

17. Lab 5 day 2 Used plates – dispose in the “contaminated waste” bags
DO NOT open plates, observe by viewing through the bottoms
Used plates – dispose in the “contaminated waste” bags P- P+ P- P+ P+ LB
LB/amp
LB/amp/ara

18. “oops” plates

20. Satellite colonies
Some cells without antibiotic resistance do become "freeloaders" and survive because other cells are doing the work of destroying the antibiotic in their immediate vicinity on the plate.
They only develop with antibiotics such as ampicillin, that are destroyed by enzymes such as beta lactamase outside of the cell.

21. Why do we get satellite colonies?
The ampicillin plate is old (meaning that the antibiotic is partially degraded) The transformed cells are plated at very high density (meaning that the plate is covered with huge number of cells) The copy number of the plasmid in the cells is so high that beta lactamase is secreted at high levels, The colonies grow on the plate for several days (allowing more time for degradation).

22. Are satellites a problem?
Probably not, provided that the colony of interest is subsequently picked and grown in fresh medium containing antibiotics.