1-15-16 Agenda & Objective

Paper Plasmid Lab

ObjectiveObjectiveCreate a model of a recombinant plasmid and use it

to explain how they are made and why they are useful

Preparation

Cut out the Cell DNA (goldenrod). These must be glued together in the order indicated at the bottom.

Keep this DNA in one long sheet.

Preparation

1. Cut out the bacterial DNA strips – pink paper

2. Toss out any 2 strips, except for the one with the replication site

3. Glue your strips together in any order, end-to-end.

4. Form a ring with them and tape the ring together.

Does everyone Does everyone have the same have the same ring of bacteria ring of bacteria DNA?DNA?

Okay…here is what we are going to do:

1. We are going to cut out the gene (DNA) for insulininsulin from some human DNA

2. We are going to cut open the DNA from a bacteria

3. We are going to fuse the 2 together4. Then the bacteria will be able to make

human insulin

What gene are What gene are we going to cut we going to cut out of the out of the human DNA?human DNA?

Where are Where are we going to we going to put this put this human human gene? gene?

WHY?WHY?

How we will do it

• We need to find the 1 1 restriction enzymerestriction enzyme that can cut BOTH DNAs for us Cuts the human DNA 2

times Cuts the bacteria DNA 1

time• Then the DNA pieces will

have the same sticky ends and will match up to each other

Human DNA

Bacteria DNA

Bacteria DNA

How many How many enzymes will enzymes will we end up we end up using to cut using to cut the DNA?the DNA?

How it’s done

• Cut the DNA from both organisms with the SAME restriction enzyme

• So they have complimentary overhanging “sticky ends”

• Combine the DNA with DNA ligase - an enzyme that will seal them together

Examples

A restriction enzyme may recognize the sequence

And it cuts after the A on both strands. What will the cut look like?

ACCGGTTGGCCA

What we use it for

• Put insulin gene into yeast to make insulin for diabetics

• Factor VIII into bacteria for hemophiliacs• Human Growth Hormone• Erythropoietin for treating anemia

Procedure

Run each enzyme slowly up and down each DNAIt must cut your PINKPINK plasmid only ONCEONCEIf must cut your GOLDENRODGOLDENROD Human DNA TWICETWICE – above and below the gene of interest.

How many How many enzymes are enzymes are we going to we going to end up end up using?using?

Procedure

Select the ONE enzyme that can cut your bacteria DNA once and your Cell DNA twice.

Record it’s name in your data table.

Use scissors to cut the DNA of the bacteria and the Cell as this enzyme would, in a staggered fashion.

Procedure

Use tape to “splice” the goldenrod cell DNA goldenrod cell DNA gene gene into your pink pink bacteria DNA.bacteria DNA.

You have now created recorecombinmbinantant DDNNAA

Analysis Questions

1. In real life, not all bacteria will successfully take up the cell DNA. Scientists need a way to select only the bacteria that have the gene. They do this by putting antibiotics on the bacteria. Those that survive must have the antibiotic resistance gene, along with the cell DNA.

What antibiotics are your bacteria resistant to?

Procedure

On your bacterial (PLASMID) DNA (the pink paper):

a. Do you have the gene for ampicillin resistance?

b. Do you have the gene for kanamycin resistance?

c. Do you have the gene for tetracyclin resistance?

d. We ALL must have the replication site

Analysis Questions

Draw in the location of the insulin gene