Laboratory #3 Transformation of Competent Cells with Foreign DNA
In the field of molecular genetics, transformation refers to the stable uptake
of foreign DNA into either a bacterial cell or a yeast cell. The types of DNA that are
most often used for the transformation are circular pieces of DNA called plasmids.
These plasmids are not circular pieces of unimportant DNA. Housed on these
plasmids are specific genes.
The process of transformation was first discovered by Fredrick Griffiths in
1928. Griffiths worked with both virulent and non-virulent strains of Streptococcus
pneumoniae (S. pneumoniae). Initially, he injected both the non-virulent R strain,
and the virulent S strain into mice. In mice where the R strain was injected, there
was no effect, whereas in the mice where the S strain was injected these mice
became sick and died. Next, if he heat killed both strains, and he injected the heat
killed bacteria, he saw no effect, suggesting that both bacterial strains were killed.
However, if he took the heat killed S strain and mixed with living (unheated) R
strain bacteria and injected this mixture into the mice, he found they got sick and
died. He postulated that whatever material was in the heat killed S strain was able
to transform the R strain from non-virulent to becoming virulent. He called this
material the transforming principle. Today, we know that the transforming principle
is DNA. As well, today we know that when the R strain is mixed with the heat killed
S strain, that the genes for virulence are being transformed into the R strain
bacteria.
Today, we have built upon Griffiths work, and have been able to develop
methods to transform foreign pieces of DNA into various different cell types,
including bacteria, yeast and even human cells. This allows scientists to be able to
perform many different experiments to shed light on important questions of human
health. When scientists transform foreign pieces of DNA into cells they use circular
piece of DNA called plasmids. These plasmids are specialized for the cell type that
they will be transformed into. For instance, plasmids that are optimized for
bacterial cells are about 3000-5000 base pairs and ones for yeast are about 800010,000 base pairs. These plasmids are not just random circular pieces of DNA. On
these plasmids are housed genes that allow the cells to survive in nutrient poor
environments or genes that confer antibiotic resistance.
Today, we will work with two different cell types. We will work with yeast,
and we will work with the laboratory bacteria E. coli (a non-pathogenic strain). The
E. coli strain we will use is called the DH5α strain, and is unable to grow in the
presence of the antibiotic ampicillin, which is placed in the agar media. We will then
transform the bacteria the pBR322 plasmid. On this plasmid is the Ampicillin
resistance gene. In your notebook, pls. hypothesize what you think will happen
upon transformation.
Additionally, we will work with a yeast strain (BY4741) that is an auxotroph.
An auxotroph is a strain that is unable to grow on media without lacking amino
acids, the building blocks of proteins, and nucleotides, the building blocks of DNA.
Specifically, this strain contains mutations in the URA3 gene, the TRP1 gene and the
HIS3 gene to render them non-functional. These genes encode enzymes that are
important in making uracil, tryptophan and histidine respectively. Therefore, if
these yeast are placed on media without uracil, tryptophan or histidine they will die.
In this laboratory, we will transform the yeast with plasmids containing the URA3
and the TRP1 and test for yeast growth on media lacking uracil or lacking
tryptophan. This process of transforming a plasmid with a functional gene to
substitute for the defective in the genome is known as complementation.
Part A: Bacterial Transformation
1. Obtain an ice bucket (ice can be found in the machine in the hall).
2. Obtain 4 tubes of competent cells (ready for transformation) from the instructor.
Pls. keep cells on ice at all times! This will affect your results.
3. Obtain 1 microfuge tube of pBR322 plasmid from the instructor
4. Obtain a microfuge tube of sterile water
5. Label the tubes in successive order, 1-4.
6. Label tube 1 control A
7. Label tube 2 control B
8. Label tube 3 pBR322 A
9. Labe tube 4 pBR322 B
10. In the control tubes, pipet 5 ul of sterile water. This serves as a control.
11. In the pBR 322 tubes, place 5 ul of pBR322 plasmid solution.
12. Mix your tubes gently, and place on ice for 20 minutes. This prepares your cells
for uptake of DNA.
13. Heat shock your cells by placing them in the 42 C bath (or block) for EXACTLY
90 seconds. The heat shock allows for your cells to take up the DNA.
14. Place cells on ice for 2 minutes.
15. Add your cells to 1 mL of LB (from instructor) and incubate your cells in the
shaking incubator at 37 C for 1 hour.
16. Plate 15 ul of your cells on both LB and LB-AMP plates as follows
a. Plate 15 ul from tube 1 on an LB plate
b. Plate 15 ul from tube 2 on an LB plate
c. Plate 15 ul from tube 3 on an LB-AMP plate
d. Plate 15 ul from tube 4 on an LB-AMP plate
17. Place in 37 C incubator overnight.
Part B-The Yeast Transformation
1. Obtain 4 tubes of yeast from the instructor-keep these cells at room temperature
2. Obtain tubes from instructor containing 40 % PEG, 1.0M LiAC (Lithium Acetate),
Sterile water, Salmon Sperm DNA, TRP plasmid and URA plasmid. Boil your salmon
sperm DNA using the hot bath in the back of the room.
3. Label the tubes of yeast 1-4
4. Label tube 1 control
5. Label tube 2 control
6. Label tube 3 URA plasmid
7.Label tube 4 TRP plasmid
8. To each tube, add 240 ul of 50% PEG.
9. To each tube add 36 ul of 1.0 ul of LiAC
10. To each tube, add 50 ul of Salmon Sperm Carrier DNA
11. To tube 3 add 34 ul of URA plasmid.
12. To tube 4 add 34 ul of TRP plasmid.
13. Place your tubes in the 30 degree block (or incubator) for 30 mins.
14. Place your tubes at 42 C for 25 mins. This heat shocks your cells, and allows for
uptake of DNA.
15. Microcentrifuge your cells for about 15-20 seconds (use quick spin setting).
Your cells will pellet to the bottom of the tube.
16. Remove the supernatant, and add about 100 ul of sterile water. Pipet up and
down to resuspend your cells.
17. Plate all the cell solution in each tube as follows:
a. Plate tube 1 on a –URA plate
b. Plate tube 2 on a –TRP plate
c. Plate tube 3 on a –URA plate
d. Plate tube 4 on a –TRP plate
18. Place in 30 C incubator for 3-5 days.