2. What are Plasmids?
Plasmids are circular pieces of bacterial DNA that often contain genes not related to basic life functions
Often contain antibiotic resistance genes
Humans often cut open plasmids…attach a desired gene…reinsert the plasmid to the bacteria
When complete, the bacteria will contain a new gene (instructions) to create a desired protein such as insulin.

3. What are restriction enzymes?
Enzymes that bacteria use to fight off viruses
Restriction enzymes are like an immune system for bacteria
They cut virus DNA at very precise locations
Virus
Virus DNA
bacteria v i u r D N A s

4. Genetic Engineering Humans are learning to manipulate DNA
We can use a restriction enzyme to cut open a bacterial plasmid… exposing sticky ends.
We would use the same restriction enzyme to cut out a human gene…exposing matching sticky ends
We can then insert the human gene into the bacterial plasmid. The sticky ends of each will match and bond.

5. So now what???
The bacteria will hopefully absorb the recombinant DNA and replicate. Each time passing the insulin gene onto its offspring.
But how do we know if the bacteria absorbs the plasmid?
Plasmids often contain antibiotic resistance genes
Ex: pKAN resists kanamycin
If the plasmid is known to resist kanamycin, the scientist will grow the bacteria in kanamycin
If the bacteria lives… it must have absorbed the plasmid.
If the bacteria dies… it did not absorb the plasmid.
Each bacteria cell now will produce insulin that can eventually be harvested for diabetics.

6. Stage 1: Prepare the plasmids to be cut by restriction enzymes
Obtain the plasmids (pKAN and pAMP)
P stands for plasmid
pKAN = plasmid with resistance to kanamycin
pAMP = plasmid with resistance to ampicillin
pKAN
pKAN
pAMP
pAMP

7. Stage 1: Prepare the plasmids to be cut by restriction enzymes
Mix plasmids with…
restriction enzymes BamH1 and Hind III…
or water K+ K- A+ A-
Bam
HinD
Bam
HinD
H2O
H2O
pKAN
pKAN
pAMP
pAMP

8. Plasmid w/ kanamycin resistance (pKAN)
Restriction enzyme: Hind III
pKAN = 5512 bp
Restriction enzyme: Bam HI
807bp restriction fragment
4705bp restriction fragment

9. There are thousands of plasmids in our microdrop sample

10. K+ (digested plasmid) + means the restriction enzymes were added
K– (uncut plasmid) - means the restriction enzymes not added
Small (807bp)
Big (4705bp)
How many plasmid fragments?
How many plasmid fragments?

11. Stage 2: Check to see if the restriction enzymes worked
DNA electrophoresis
Plasmid fragments are loaded into a gel
Connected to a power supply
Separates fragments based on their sizes
Smaller fragments travel further through the gel

12. We will then micropipette the plasmids

13. Load the plasmids into an electrophoresis chamber

19. Connect the electrophoresis to a power supply…DNA has a negative electric charge.