4-2 Sources of DNA

Background• Nature = DNA in cells • Lab= Manipulate and grow cultures of

different cells to access DNA– Grow in medium– Collect and break open cells (lysis)– Separate and isolate DNA

Genetic code is the same from organism to organism BUT the packaging and locationOf DNA varies among organisms.

Bacterial DNA• No nucleus or organelles (except ribosomes)• 1 long, circular DNA (supercoiled like twisted

rubber band)• ~1000 genes• “Transformation” can transfer genetic

information between bacterial cells (exchange plasmids)

• Plasmid = extra ring of DNA in cytoplasm found in some bacteria– 5-10 genes – Codes for proteins that might be needed under

extreme conditions • R-plasmid = antibiotic resistant genes

Bacterial Cell Cultures

Bacterial Cell Cultures1) Solid medium (agar plates)• Agar (algae powder) is mixed with water• Heat until suspended• Autoclave (high temp/high pressure)• Agar solution cooled to 65˚C• Pour into sterile petri dishes using sterile technique• Agar solidifies 15-20 minutes• Streak the bacteria on the plate using the “triple Z

streaking method”

Autoclave= instrument that creates high pressure and high temperatures

to sterilize equipment and media

What are you looking at in the lab?

• If you want the DNA then you must EXTRACT it from the bacterial cell by breaking open the cell (lyse)

• If your product is a protein, you must also EXTRACT the proteins by lysing the cell

• Bacterial cells are very easy to grow but must lyse cells to get products.

Bacterial Cell Cultures2) Liquid medium (broth)• LB powder base is mixed with water • Autoclave (high temp/high pressure)• Introduce bacteria to the broth

(inoculate) using sterile technique• Shake cultures so bacteria can grow

Eukaryotic DNA• DNA from protists, plants, fungi and

animal cells• Packaged into Chromosomes– Single long linear DNA coiled around

histone proteins– Each single DNA may contain several

million or more nucleotides and up to many thousands of genes

– Same genetic code (4 nitrogenous bases) as bacteria just more genes

Mammalian Cell Cultures• More challenging to grow than

bacteria• Mammalian cells depend on other

cells for products and stimuli for growth

• Cells are grown in a liquid broth in special tubes and bottles with a bottom surface to which the cells can stick

T-Flask

Roller Bottles

Mammalian Cell Cultures• Media contains special nutrients

(sugars, salts, proteins) ideal for each type of cell being grown– Special indicators can be added to

monitor growth • Phenol Red – pH indicator. Turns

yellow/gold as solution becomes acidic (too much carbon dioxide and other waste products) = cells overgrowing!

What are you looking at in the lab?• If you want the DNA, then you must EXTRACT it

from the mammalian cell by breaking open the cell (lyse)

• If your product is a protein, you can EXTRACT it from the growth media– Mammalian cells release their proteins from the cell

into the growth media so its easy to harvest.– Cells are unharmed!– Mammalian cells hard to grow but easy to harvest

protein products!

Extra Stuff you might like to know(Dr. Karkare supplements)

Characteristics of Mammalian Cells

• Require stringent environment, very shear sensitive • Many are attachment dependent• In addition to typical metabolic requirements, cells need growth factors

and other proteins• Mammalian cells grow at a much slower rate than bacteria and yeast,

doubling every 14 to 70 hours• Cell growth can be described by lag, exponential growth, then stationary

and death phases

Cell Culture Musts

• Aseptic technique– Laminar flow hoods– Minimize air flow disturbance– Sterile plastic/glassware– Use of sterile gloves etc. (humans are primary

contamination source!)– Minimize exposure of culture to open air

Cell Culture Musts

• Appropriate culture environment– Culture media containing glucose, amino acids,

serum (FBS or appropriate serum substitutes), lipids etc.

– Incubation at 37o C– pH control with bicarbonate buffer (CO2

incubator)

T-Flasks•T-Flasks: Flat sided plastic flasks used for small scale culturing (25 cm2 to 175 cm2

•Surface treated to generate highly charged oxygen ions on the plastic creating negative charges which attract most cell lines•Other methods use chemical coatings such as poly D-lysine

Automated Cell Culture machine at ATCC