AP Biology

Biotechnology Part 3

Bacterial Cloning Process

Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

Restriction Enzymes & Sticky endsRestriction 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 PlasmidThe 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 PlasmidThe 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)