1. Bacterial Transformation
Lab 6A

2. Our Goals Insert foreign gene into plasmid
Insert recombinant plasmid into bacteria
Grow transformed bacteria

3. Questions We Need Answered
Did the bacteria take up the plasmid?
If so, did the gene of interest get inserted into the plasmid?
Analyzing our plates at the end of the lab will answer these questions and tell us if we achieved our goals

4. What must you know to understand this lab?
Essential Knowledge
What must you know to understand this lab?

5. Antibiotic Resistance
Resistant bacteria can survive in the presence of antibiotics
We will be using ampicillin
Our plasmid is resistant to ampicillin

6. Lac-Z gene Lactose = glucose + galactose
Lactose is broken down by the Lac-Z gene
Lac-Z will also digest similar compounds

7. X gal X gal is a compound that is similar to lactose
X gal turns blue when it is digested by the Lac-Z gene

8. Ampicillin Resistance
Engineered Plasmids
We will be using pBlue
Lac-Z
gene
Ampicillin Resistance
gene

9. Restriction Enzymes
Our plasmid DNA and our foreign DNA arrive pre-cut by the same restriction enzyme
The restriction enzyme made a single cut in the plasmid
The restriction site is within the lac-Z gene

10. Ampicillin Resistance
Restriction site
Lac-Z
gene
Ampicillin Resistance
gene

11. We will be using 3 types of agar
LB + amp
LB + amp + X gal

12. Each Group Will Run Six PlatesLB
- plasmid LB
+ plasmid
LB/amp
- plasmid
LB/amp
+ plasmid
LB/am/X-gal
- plasmid
LB/am/X-gal
+ plasmid

13. How will we know if we are successful?

14. LB Plates There is nothing in the LB agar to interfere with growth.
Growth on both the + and – plates indicates the bacteria are alive and well.
A “lawn” is too many colonies to count
- plasmid
+ plasmid

15. What if nothing grows on either LB plate?

16. LB/amp Plates
Only bacteria that have taken up the plasmid will grow on the plates with ampicillin
How do you know?

17. If the plasmid is inserted into the bacteria,
it will become antibiotic resistant and will grow on the ampicillin plate
only transformed bacteria grow
LB + amp

18. LB/amp Plates
No growth on – confirms ampicillin is working because bacteria without the plasmid are not resistant and die
Bacteria growing on + contain the plasmid and are amp resistant
- plasmid
+ plasmid

19. LB/amp Plates
Would you expect isolated colonies or a lawn on the + ? Why?

20. LB/amp Plates What if your – shows unexpected growth?
Can you tell by looking at + which ones are recombinant?

21. LB/amp/X-gal
Color of colonies on X-gal plate will tell you if gene of interest has been inserted into the plasmid or not
Blue colonies indicate Lac Z gene is digesting X-gal
White colonies indicate Lac Z gene in not functioning

22. LB/amp/X-gal
If ends of lac Z gene come back together and are resealed by DNA ligase during the transformation process, the Lac Z gene will be functional and will turn X gal blue.

23. LB/amp/X-gal
If the gene of interest is successfully inserted between the free ends of the plasmid, the Lac Z gene will be “broken”.
If the Lac Z gene is interrupted, X-gal will not be digested and the colonies will remain white

24. If the foreign DNA is inserted into the plasmid,
The Lac-Z gene will be interrupted and bacteria will stay white
recombinant plasmid
amp
resistance
“broken” LacZ gene
inserted gene of interest
LB + amp + X gal

25. Blue vs. White on X-gal Plate
Bacteria take up plasmid
Functional LacZ gene
X gal is being digested
Bacteria make blue color
Bacteria take up recombinant plasmid
Non-functional LacZ gene
X gal is NOT being digested
Bacteria stay white color

26. What if you just see blue colonies on + ?
Should you ever see blue colonies on - ?
Should you see isolated colonies or a lawn on the X gal plate? Why?
- plasmid
+ plasmid

27. What do we really want to see?

28. White Colonies on X-gal !
LB + amp + X gal
SUCCESS!

29. Any
Questions?