1. Transformation of Escherichia coli
Exercise 9

2. Experiment Goals
Understand the transformation procedure using the heat shock method.
Understand how DNA can be transferred to an organism and the change in phenotype that may result from such a transfer.
Transformation of a gene for resistance to the antibiotic ampicillin into a bacterial strain (E. Coli) that is sensitive to ampicillin.

3. Bacterial Transformation
Definition:
Transformation is the genetic alteration of a cell resulting from the uptake and expression of foreign genetic material (DNA). i.e. the act of putting foreign DNA into a bacterial cell
Occurs in nature, but rarely
If the foreign DNA has an origin of replication recognized by the host cell DNA polymerases, the bacteria will replicate the foreign DNA along with their own DNA.
The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated

4. Escherichia coli (E. coli)
Lives in the human gut
Relatively simple and well understood organism
Its genetic material consists of:
DNA 3-5 million base pairs in length,
Plasmids, usually ranging from 5,000-10,000 base pairs in length
It is these plasmids that bacteria can transfer back and forth, allowing them to share genes among one another and thus to naturally adapt to new environments.

5. Plasmids
Plasmids
small
circular
extrachromosomal DNA
Self-replicating DNA molecule separate from chromosome

6. Plasmids
Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival.
In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes.
This natural mechanism allows bacteria to adapt to new environments.
The occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.

7. Transformation Applications

8. How ? Use enzymes to cut plasmid DNA
Insert a DNA sequence for a desired protein
Insert plasmid into bacteria and grow…grows fast!
Isolate protein made by bacteria

9. Competency
Not all bacteria take up free-floating DNA in the environment
Bacteria which are able to uptake DNA are called "competent"
Some bacterial strains have NATURAL COMPETENCE. Ex. Bacillus, Streptococcus, Haemophilus and Neisseria.
Some bacterial strains, such as E. coli, can be made ARTIFICIALLY COMPETENT.

10. Artificial Competency
Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane.
In order to make bacteria take in the plasmid, they must first be made "competent" to take up DNA.
This is done by creating small holes in the bacterial cells by different methods.
Cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth.

11. Experiment
In this experiment, we will introduce a gene for resistance to the antibiotic ampicillin into a bacterial strain that is sensitive to ampicillin
If the sensitive bacteria incorporate the foreign DNA, they will become ampicillin resistant.

12. Sterile Technique
It is important not to contaminate E. Coli plates with external bacteria from hands or the table, etc
The plates without ampicillin are especially prone to contamination, since anything can grow on those plates.
Wearing gloves can help avoid contaminating plates and solutions
Keep the lids on the plates whenever not adding things to the plates
All of the solutions and plastic ware will be using have been sterilized

14. Steps of Bacterial Transformation
Competency
Transformation
Recovery period
Growth & Isolation

15. 1- Competency Label two sterile microtubes: one “+”and the other “-”.
Using a disposable pipette, add 250 μl of 50mM CaCl2 solution to each tube (“+” and “-”) and place them both on ice.
The cells are kept cold to prevent them from growing while the plasmids are being absorbed

16. 1- Competency
Use a sterile plastic loop to transfer one or two 3 mm bacterial colonies to the “+” tube, return tube to ice (Do not pick up an agar as it may inhibit the transformation process)
Transfer a mass of cells to the “-” tube
Add 10 µl of ampicillin resistant plasmid directly into the CaCl2 in “+” tube.
Return the “+” tube to the ice. DO NOT add the plasmid to your "-" tube. Incubate both tubes on ice for 15 minutes.

17. 1- Competency
                                                                                                                            
Ca2+ neutralizes the negative charges on the phosphate backbone of the DNA, so that the DNA is not repulsed from the phospholipids of the cell membrane.
This allows the DNA to more easily pass

18. 2- Transformation
Insertion of a plasmid that contains a gene for the resistance to ampicillin
Heat shock - helps plasmid enter the cells

19. 2- Transformation

20. 3- Recovery period
Allow the cell to regain strength and start to multiply
Necessary to allow expression of the antibiotic resistance gene on the plasmid
Add 250 µl sterile Luria Broth to both tubes using a sterile pipette
Move tubes to water bath at 37oC for 5 min.

21. 4- Growth & Isolation
Mark four Luria Broth agar plates

22. Transformation procedure
Plasmid
CaCl2
E. coli
E. coli +
cold!
pAmp
42oC
Recover
at 37oC
E. coli
LB + Amp
Incubate at
37oC overnight
LB + Amp

23. 4- Growth & Isolation

24. Transformation Efficiency
After incubation count the colonies on each plate.
Calculate the transformation efficiency
Transformation Efficiency = no. of colonies observed/ µg Plasmid

25. Plasmid DNA spread in μg = A x B
A. Determine the Total Amount of plasmid DNA
(DNA in μg) = (concentration of DNA in μg/μl) x (volume of plasmid in μl)
Determining the fraction of plasmid DNA (in the bacteria) that actually got spread onto the LB/amp plate
Fraction of Volume spread on LB/amp plate (μl)
DNA Used = Total sample volume in test tube (μl)
Plasmid DNA spread in μg = A x B