1. Bacterial Transformation
AP Biology Transformation Lab

2. What is Transformation?
Changing the genes and phenotype of a bacteria by uptake of foreign/new DNA
a natural process that bacteria have evolved in order to obtain DNA from their environment.
enables scientists to insert genes by recombinant techniques and place the plasmid into a bacteria for expression

3. What is Transformation?
In order to transform bacteria we need to overcome two problems
Disadvantage –cells that contain plasmids grow more slowly
There is pressure on cells to get rid of their plasmids
Needs to be an Advantage to keep plasmids
Antibiotic resistance
How do we tell which cells have the plasmid?
We use a marker
Grow the bacteria on plates that contain the antibiotic
Use a color pigment marker that is present when a particular enzyme is present
Let’s review bacterial DNA first…

4. Let’s Review: Bacterial genome
Bacteria are prokaryotes—no nucleus.
The area where DNA is located is called the nucleoid
DNA is organized in one double stranded circular molecule

5. Bacterial DNA
Bacterial cell
Plasmid DNA
Genomic DNA

6. Size

7. Which bacteria will we be using?
Escherichia coli is the most common bacterium in the human gut. It has been extensively studied in the laboratory and is an important research organism for molecular biology.
E. coli reproduce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight.
Like all bacteria, E. coli has no nuclear envelope surrounding the bacterial chromosome and thus no true nucleus.
All of the genes required for basic survival and reproduction are found in the single chromosome.

8. What is a Plasmid? A circular piece of autonomously replicating DNA
exists outside the main bacterial chromosome
Originally evolved by bacteria
Carries separate genes for specialized functions.
In genetic engineering, plasmids are one means used to introduce foreign genes into a bacterial cell.
Scanning electron micrograph

9. What is carried on the Plasmid?
The plasmid contains genes necessary for survival and can be passed from one bacteria to another
ampR gene
confers resistance to the antibiotic ampicillin.
E. coli cells containing this plasmid, can survive and form colonies on LB agar that has been supplemented with ampicillin.
Cells lacking the ampR plasmid are sensitive to the antibiotic, which kills them.
An ampicillin-sensitive cell can be transformed to an ampicillin-resistant cell by its uptake of a foreign plasmid containing the ampR gene.
The same can be said for the lac gene, which codes for lactose. I
If this gene is taken in, the organism can break down lactose.

10. Transformation has 4 main steps
Prepare cells
Make them competent
Incubate with plasmid
Plasmid associates with membrane of cells
Shock the cells
To initiate plasmid uptake
Allow cells to recover and plate on agar
Allow cells to recover in rich medium for 0.5 – 1 h (without antibiotic to avoid stressing the cells)
Plate on agar with antibiotic for selection
Next day can pick single colonies or clones

11. The Transformation Lab…
Our plasmid: pBlu plasmid
Into E. coli (scary?…no!)
Our plasmid contains genes for:
AMP= ampicillin (an antibiotic) resistance
Beta-galactosidase-an enzyme that converts X-Gal  Indo Blu
RNA
Protein that allows for antibiotic resistance
RNA
Enzyme that breaks down X-Gal to make Indo Blu

12. How do we get the plasmid inside of the bacteria?
To transform cells, you first need to make them competent to take up extracellular DNA.
Obtain E. Coli bacteria cells + Add to ice cold CaCl2
Helps plasmid attach to bacteria
Makes the cell competent
Add plasmid to same microtube
1. E. Coli
2. pBlu plasmid

13. How It Works Transformation solution CaCI2
Positive charge of Ca++ ions shields negative charge of DNA phosphates
DNA becomes “neutral” and can pass through the cell membrane
Ca++ O P O
Base O O
CH2
Sugar O
Ca++ O P O
Base O O
CH2
Sugar OH

14. How do we get the plasmid inside the bacteria?
Wait…and then
Heat shock! This temporarily opens pores to allow the plasmid to enter the bacteria…timing is critical!!!

15. Chemical transformation
Ice-cold CaCl2
To make competent
Slows the fluid cell membrane
Heat shock
Increases permeability of membranes by opening pores
Plasmid DNA is taken up
Nutrient broth incubation
Allows beta-lactamase expression
Bacterial
chromosomal DNA
Cell wall
GFP
Beta lactamase
(ampicillin resistance)
pBLU plasmids

16. What is Nutrient Broth? Luria-Bertani (LB) broth
Medium that contains nutrients for bacterial growth and gene expression
Carbohydrates
Amino acids
Nucleotides
Salts
Vitamins

17. How will we know if the bacteria actually got into the plasmid?
Any ideas?
We can grow the bacteria on a plate:
That contains ampicillin and X-Gal
Regular bacterial medium
What do you predict will happen in each?

18. Predict pBlu Control What will we observe??? Amp X-Gal Regular Amp

19. Growing the bacteria After they have received the plasmid…
Place on a growth media and allowed to grow.
spread evenly
discrete colonies
(~106 cells)
~ 100 µl
overnight
37 ºC