1. The following may contain violence and sexuality.
Viewer discretion is advised.

2. Transformation of E.coli with pGal
The best of the blues
Ampicillin resistance is not futile
But only if you are competent

3. Bacterial structure
E.coli is a bacteria that is normally found in your body. Bacteria has DNA in2 forms: a giant loop that contains most of the bacterialDNA and is attached to the cell membrane and a small loop of DNA called a plasmid

4. Conjugation of bacteria
Under certain conditions these plasmids can move from cell to cell. And carry a gene from one bacteria to another and teach it new tricks. It is a form of
Sexual reproduction
in bacteria and happens
Under extreme conditions

5. Genetic engineering of plasmids
Because plasmids can go in and out of cells we can genetically engineer the plasmid to contain a certain gene.eg human insulin, or in this case ampicillin resistance and beta -galactosidase

6. Engineering a plasmid First you use a restriction enzyme
to cut the gene you want from a
piece of DNA.
Use PCR to make lots of copies
of the gene
Use the same restriction enzyme
to cut open the plasmid.
Then you splice them together
At their sticky ends with ligase

7. Competancy E.coli doesn’t readily take up a new plasmid.
In order to get the bacteria to take up the plasmid it must be competent. (we have to mimic the extreme conditions and make the membrane and wall porous).
We put it in a salty environment (CaCl2)
We shock it with hot and cold temp.
Kind of like having dried, cracked skin in the winter.
Then the E.coli are fragile so you have to let them recover.

8. Competancy

9. Transformation Once the bacteria has been made competent
It is mixed with the engineered plasmids.
Some of the bacteria will take them into the cell.
Now the bacteria can make a new protein. It is transformed.

10. Your lab Your cleverly engineered plasmid is called pGal
It has 2 regions of interest:
Ampicillin resistance (it makes an enzyme that it exudes . It diffuses into the gel and cuts up the ampicillin so it doesn’t work
Beta-galactosidase ( an enzyme that cuts all the galactosides X-Gal then the
bacteria turns blue)
Only the transformed cells
will grow and turn blue

11. What you have to do Read your lab carefully before class
Make the bacteria Competent
Make a control with no plasmid
Make a variable with added plasmids
Make the 3 agar plates
Control1 X-Gal (food only)
Control 2 X-Gal + Amp ( food and antibiotic only)
Experiment +DNA X-Gal+amp + pGal ( food and antibiotic in agar plate

12. Read the lab carefully before next class
Add control bacteria with no plasmids to control plates 1 and 2
Add the transformed bacteria with plasmids to plate 3
Allow incubation
Count the # of transformed colonies
Calculate the bacterial transformation efficiency.
Read the lab carefully before next class