Transcript Plasmid w/ kanamycin resistance (pKAN)

What are Plasmids?
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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.
What are restriction enzymes?
• Enzymes that bacteria use to fight off viruses
Virus
Virus DNA
vi u
r s DNA
bacteria
• Restriction enzymes are like an immune
system for bacteria
• They cut virus DNA at very precise locations
Genetic Engineering
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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.
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.
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
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
H2O
Bam
HinD
H2O
pKAN
pKAN
pAMP
pAMP
Plasmid w/ kanamycin resistance
(pKAN)
Restriction enzyme:
Hind III
pKAN = 5512 bp
Restriction enzyme:
Bam HI
4705bp
restriction
fragment
807bp
restriction
fragment
There are thousands of plasmids in our
microdrop sample
K+
(digested plasmid)
K–
(uncut plasmid)
+ means the restriction enzymes were
added
- means the restriction enzymes not
added
Big (4705bp)
Small
(807bp)
How many plasmid fragments?
How many plasmid fragments?
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
We will then micropipette the plasmids
Load the plasmids into an electrophoresis chamber
Connect the electrophoresis to a power
supply…DNA has a negative electric charge.