Recombinant DNA

Restriction Enzymes• Enzymes that break down DNA by cutting it at

a specific site (restriction site)• Originally used as cellular defense against

invaders• Used in Biotech to create recombinant DNA or

for DNA fingerprinting• Each enzyme has a unique restriction site that

it recognizes

Example of Restriction Sites

• EcoRI5’ GAATTC 3’3’ CTTAAG 5’

5’ G AATC 3’3’ CTTA G 5’

“Sticky ends” (sections of single stranded DNA) are created.

Time to Practice

• DNA Scissors Activity will enable you to visualize how restriction enzymes work!

What is Recombinant DNA?

• DNA that contains a gene or genes from more than one organism

What are some uses for recombinant DNA?

• Create GMO’s– Protein production– Organisms with specific traits

How do you create recombinant DNA?

• Identify desired gene and cut it out with restriction enzymes. (sticky ends)

• Cut a plasmid with same restriction enzyme.

• Join your gene to plasmid and seal with ligase to form a recombinant plasmid

• Put recombinant DNA into a host cell to replicate.

• Detect cells that have recombinant DNA and can make the desired product.– Example – antibiotic resistance or ability to glow

How does this relate to class?• Using recombinant plasmid pGLO in transformation

lab• Contains Green Fluorescent Protein gene (GFP)

that glows green in UV light and is ampicillin resistant

Why is this important in the real world? (not just in science class)

• Real uses of recombinant DNA?• Ethical issues?

Time to Practice!

• We are going to practice creating recombinant DNA using paper plasmids. If you master this, you will get to make real recombinant plasmids during our transformation lab.

Transformation Lab Vocab• Sterile Technique– Proper way of handling bacteria to avoid

contamination.

• Streaking– Process of spreading bacteria on an agar plate to

separate the cells

• Agar– Substance on plate that provides support and food

for bacterial growth

• Arabinose– Added to Agar plates, sugar that’s food for bacteria

• Ampicillin– Added to Agar plates, Antibiotic that kills bacteria

• Bacterial Library – A collection of bacterial cells that contain recombinant

plasmids with random DNA inserts from a single species

• Colony – A clump of genetically identical cells growing on an agar

plate (clones)

• Cloning – When a population of cells is prepared by growth from a

single cell (entire population is genetically identical)