1. Do Now What is E.coli? And why is it used for biotech?
What have you learned so far in this biotech unit?

2. What is the evolutionary purpose for plasmids?
Do Now
What is the evolutionary purpose for plasmids?
What are aseptic techniques?

3. Objectives
SWBAT:
Using the biotechnology skills developed so far, get the pARA-R plasmid containing the rfp gene into bacterial cells
Get those cells to express the rfp gene and make the mutant fluorescent protein
Explain the necessary biological processes to make this change happen

4. Transforming E. coli with a Recombinant Plasmid
Laboratory 5

5. Introduction Transformation Process of taking up foreign pieces of DNA
In this case, a plasmid
Usually happens through conjugation
Not very common in mature!
Can occur under experimental conditions (1 cell in a thousand!)
Advantages include antibiotic resistance
All cells that undergo binary fission after insertion of plasmid will possess it:

6. Plasmid DNA Insertion

7. Transgenic Colony Allowed to Grow

8. Introduction Transformation efficiency: Size of plasmid
Large
Less likely to take up plasmid
Must pass through plasma membrane and cell wall
Small
More likely to pass through
Shape of plasmid
Supercoiled easiest to pass through
Nicked-circle or mulitimer harder to get through
Most likely your tubes will contain

9. Introduction What are competent cells?!?! Need to “heat shock” cells
Cells ready to receive plasmids through lab procedures
Soaked in calcium chloride
PM and DNA negatively charged
Calcium ions (Ca++) neutralize charges to allow for plasmid to pass through PM
Need to “heat shock” cells
Creates pressure differences
Cold then hot
Then feed and recover!!

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

11. Transforming Escherichia coli with pARA-R
Bruce Wallace
Competent Cells
pARA-R
Recombinant Plasmids

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

13. Introduction Spread cells on various plates (3) LB plate LB/amp plate
Only bacterial food
LB/amp plate
Contains ampicillin
Antibiotic prevents bacteria from forming CW
Cells that contain ampr produces protein that destroys ampicillin
Cells will grow!
LB/amp/ara plate
Contains arabinose
Needed to express the rfp gene
If take up plasmid, this helps with transcription of gene

14. Materials Reagents and Cultures Equipment and Supplies pARA-R tube
100 µL competent cells (LMG)
350 µL LB broth (sterile)
Crushed ice in Styrofoam cup
Sterile agar plates LB
LB/amp
LB/amp/ara
Equipment and Supplies
P-20 micropipette and tips
P-200 micropipette and tips
42oC water bath
1 pack sterile spreaders
Plastic microfuge tube rack
1.5 mL microfuge tubes
Marking pens
Disinfectant spray
Cybersafe

15. What will you need to do? Discuss proper aseptic techniques
Protect themselves
No contamination!
Assign tasks….
Turn on water bath the day before to check temperature

16. What will you need to do?
KEEP COMPETENT CELLS FROZEN until transformation day
Day of lab:
Take out number of tubes need for transformation
Each group will need 100 µL of cells
Each tube 500 µL of cells
Place tubes in wet ice to defrost
Will take about minutes
Resuspend cells prior to aliquoting
Gently pump in and out with P-200 pipette
Return any unused tubes with cells immediately to Styrofoam chest and place into freezer

17. What will you need to do?
Cells need to be in contact with ice or water during exercise
Push tubes to bottom of foam rack
Have cell-contaminated waste bag
Deposit tips, tubes, and spreaders that have come in contact with bacteria
Bag will be autoclaved after use

18. Methods….diluted version
2 clean microfuge tubes – KEEP PIPETTE TIPS SEPARATE!!!! P+ P-
Label BOTTOM of plates (agar side) near edges
After step 12, let rest for a few minutes at room temperature
Step 14d
Clamshell opening
GLIDE spreader, do not dig
Be sure to invert the plates when incubate
Decrease condensation and skewed results

19. Predictions…… P+ P- P+ P- P+ LB
LB/amp
LB/amp/ara

20. Predictions…… P+ P+ P- P- P+ + + + - + LB
LB/amp
LB/amp/ara

21. Conclusions 2 tubes P+ contains E. coli and plasmid
P- contains only E. coli

22. Growth of transformed bacteria on various plates
Bruce Wallace
P+ plates LB
LB/amp
LB/amp/ara
P- plates
No growth LB
LB/amp