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What are Plasmids?
• Plasmids are circular pieces of bacterial DNA that often contain genes not related to basic life functions
• Often contain antibiotic resistances
• Humans often cut open plasmids…attach a desired gene…reinsert the plasmid to the bacteria
What are restriction enzymes?
• Enzymes that bacteria use to fight off viruses
• Restriction enzymes are like an immune system for bacteria
• They cut DNA at very precise locations
bacteria
Virus
Virus DNA
v i ru s D N A
Genetic Engineering• Humans are learning to manipulate DNA• We use restriction enzymes to cut open a
bacterial plasmid…• We use the same restriction enzymes to cut out
a human gene…• Once removed, we can insert the human gene
into a bacterial plasmid
Insulingene
Insulingene
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 pAMPpKAN pAMP
Stage 1: Prepare the plasmids to be cut by restriction enzymes
• Mix plasmids with…– restriction enzymes BamH1 and Hind III…– or water
pKAN pAMP
BamHinD
BamHinD
pKAN pAMP
H2O H2O
K+ K- A+ A-
Restriction enzyme: Hind III
(cuts @ bp 234)
Restriction enzyme: Bam HI
(cuts @ bp 2095)
Plasmid w/ kanamycin resistance (pKAN)
pKAN = 4194 bp
1861 bp restriction fragment2333 bp
restriction fragment
K+(digested plasmid)
+ means the restriction enzymes were added
K–(uncut plasmid)
- means the restriction enzymes not added
How many plasmid fragments? How many plasmid fragments?
Small (1861bp)Big (2333bp)
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
10,000bp
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
1,500bp
1,000bp
Electrophoresis: sizes DNA fragments
Kno
wn
DN
A
mar
kers
K+
K-
A+
A-
2,000bp
500bp
Look at the lab handout and let’s predict the A+ fragments
How many marks will appear in the K- and A- lanes?
What just happened? Why not just 1 band?
10,000bp
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
1,500bp
1,000bp
Mar
ker
DN
A
K+
K-
A+
A-
2,000bp
500bp
Mar
ker
DN
A
K+
K+
K+
A+
A+
A+
2011 Class Data
M A+ K+ A+ K+ A+ K+ A+ K+ K-A- M
500
1000
1500
2000
300040005000
10,00080006000
500
1000
1500
2000
300040005000
10,00080006000
So now what???
• Plasmids have been engineered for human uses. The human gene for insulin (red) can now be added to the plasmid. The bacteria will produce insulin for diabetics!
So now what???
The bacteria with the recombinant DNA replicates, thus passing the insulin gene onto its offspring. Each cell now will produce insulin for humans to harvest and use.
10,000bp
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
1,500bp
1,000bp
Mar
ker
DN
A
K+
K-
A+
A-
2,000bp
500bp
Mar
ker
DN
A
K+
K+
K+
A+
A+
A+
10,000bp
8,000bp
6,000bp
5,000bp
4,000bp
3,000bp
1,500bp
1,000bp
Mar
ker
DN
A
K+
K-
A+
A-
2,000bp
500bp
Mar
ker
DN
A
K+
K+
K+
A+
A+
A+