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

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

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

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

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

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.

Competancy

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.

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

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

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