Download Biotechnology II PPT

AP Biology
Biotechnology Part 3
Bacterial Cloning
Process
Bacterium
Gene inserted into
plasmid
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
Recombinant
bacterium
Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest
Protein expressed
by gene of interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Restriction Enzymes & Sticky ends
Restriction enzymes are special
enzymes, found in bacteria, that cut
DNA at special places.
The specific place that the DNA is
cut is called a restriction site.
When the restriction enzyme cuts
the DNA at the restriction site, it
creates fragments of DNA called
restriction fragments.
Restriction fragments have “sticky
ends” that can match up with the
ends of other fragments.
Cut the DNA and Bacterial Plasmid
The first step in bacterial cloning is
to use specific restriction enzymes
to cut the DNA and bacterial
plasmid. This will cut both at the
same place!
Once the DNA has been cut, you
can mix the fragments together and
allow them to be joined  using
ligase.
Reintroduce the Plasmid
The next step is to reintroduce the
recombined plasmid back into the
bacteria. This is done through
transformation  bacteria picking up
foreign DNA.
The new bacteria are called a
cloning vector. A vector is an
organism that carries a recombined
plasmid.
Let the Bacteria grow and reproduce
You will know let the bacteria grow and
reproduce. The recombined bacteria will
grow through binary fission and create
new bacteria that should have the
plasmid.
Identify the transformed bacteria and culture
the experimental bacteria
Once you have grown the recombined
bacteria, you will now need to isolate the
experimental bacteria.
1. You will create a radioactive nucleic acid
probe using phosphorous.
2. Then you will denature the DNA using
heat to expose the bases.
3. The radioactive probe will join with the
complimentary bases on the gene of
interest.
4. Use a special film that will show the
radioactive colonies and separate these
from the others.
Hershey-Chase Experiment
Grow, verify protein production,
and then use
Source of the DNA for Transformation?
Scientists must go from mRNA
back to DNA to make the process
easier. This is a tough thing to
do because:
- Prokaryotic DNA does not
have introns.
- Modified mRNA must be
collected after it leaves the
nucleus and turned back into
DNA.
- Use reverse transcriptase to
turn single stranded RNA into
double stranded DNA. You
will need to first add a
promotor sequence.
- New cDNA (complimentary
DNA) can be stored for use.
INTRONS
Yeast Artifical Chromosome (YAC)