Download Paper Plasmids Lab

Recombinant Paper Plasmids Lab
Some of the most important techniques used in biotechnology today involve
making recombinant DNA molecules. A recombinant object has been reassembled
from parts taken from more than one source. Your genome is recombinant in that
part of ,it came from your mother and part came from your father. Recombinant
DNA molecules are pieces of DNA that have been reassembled from pieces taken
from more than one source of DNA. A gene coding for particular protein is
transferred into a host organism. The host multiplies, and produces the desired
protein in volume. For example~ the gene that codes for the production of human
insulin has been inserted into the common bacterium, E. coiL Often, one of these
DNA sources is a plasmid.
A plasmid is a small, circular DNA molecule that can reside in cells,
particularly bacteria. The cell's DNA replication enzymes copy them because they
contain a special sequence of DNA bases called an origin of replication. DNA
replication enzymes assemble at this special sequence to begin synthesizing a new
DNA molecule. Replication origins are essential to heredity; if a DNA molecule does
not have a replication origin, it can not be copied by the cell and wi" not be
transmitted to future generations.
P!asmids often contain genes for resistance to antibiotics. Antibiotics are
natural substances produced mostly by soil microbes. Antibiotic production allows
these microbes to kill of competing microorganisms. Scientists use antibiotic
resistance as a marker to select for particular cells.
In this experiment, you will construct a bacterial plasmid using paper. You
will locate and map various important sites, such as the replication origin, antibiotic
resistance genes, and restriction enzyme recognition sites (nucleotide sequences
on the DNA which the enzyme uses to it), on the plasmid. Then you will construct
a section of "host cell" DNA, identify restriction enzyme recognition sites on it, and
cut the DNA with staggered cuts, producing sticky ends. You will cut open the
plasmid and insert the host cell gene into the plasmid, producing a "recombinant
paper plasmid."
By simulating the production of a recombinant plasmid, you wHlunderstand
how they are produced, information you wi" need for the next lab.
Materials:
Paper models of plasmid DNA, Cell DNA,
Acetate (transparency) models of Enzymes
Scissors
Tape
Methods:
1. Cut out the paper and acetate models along the heavy dotted lines. Do not throw
away the "key," which indicates what the hatch marks stand for.
2. Tape the plasmid strips end to end, IN SEQUENCE, to form a circle. Have the
printed bases facing outwards.
3. Using a pencil or colored pencils, mark the locations where the eight restriction
enzymes can cleave the plasmid. For example, use the enzyme HindIII. By.
moving the acetate card along the plasmid, you will find the sequences where
cleavage can occur. Be sure to identify the enzyme name (with pencil or colored
pencil)at the location on the plasmid DNA.
4. Using a whole piece of plain white paper, diagram a "map" of the plasmid. Draw
a large clrcle; mark the replication origin at "12:00," (You do not recopy all the
bases; just draw symbols.) Also mark the restriction sites and antibiotic
resistance sites. Label this as "Original Plasmid,"
5. Tape the strips of "Cell DNA" end to end, in order from #1 to #6, to form a long flat strip. Locate the protein gene (the marked section), 6. The object is to cleave the protein gene out of the cell DNA and insert it into the
plasmid, Test the restriction enzymes on the cell DNA; identify the enzymes
which will cut the DNA OUTSIDE the protein gene, If an enzyme cuts INSIDE the
gene, you cannot use it. Then identify which of the helpful enzymes will also cut
the plasmid open, so that when both the cell DNA and the plasmid are cleaved,
the exposed sticky ends are complementary to each other.
7. Cut your plasmid and cell DNA as indicated by the enzyme, making sure to cut
BETWEEN the nucleotide chains as the enzyme does. Use tape to join the cell
DNA to the plasmid by matching the exposed ends to one another.
Note: You CAN cut out pieces of your plasmid, making the final plasmid smaller
than if you include all of its DNA, The smaller the plasmid, the better it is
absorbed by the host bacteria. BUT, you must keep the replication origin and at
least one antibiotic gene.
8, Draw another plasmid map, on a different sheet of paper, Show the inserted
protein as well as the other sites, Clearly identify the restriction enzymes used to
splice the plasmid. Label this as "Recombinant Plasmid."
9. Attach the plasm ids and maps to your lab report.
Questions:
1. Why must you use the same restriction enzyme on the cell DNA and on the
plasmid?
2. What is the importance of DNA ligase enzyme?
3. Locate the protein gene. The genetic code is on the left side, reading top to
bottom. (5' to 3'.) What is the first mRNA codon in this sequence? What is the
amino acid coded for by this codon?
4. What would the last mRNA codon be for this gene? What type of codon is this?
5. USing your recombinant plasmid, which antibiotics could be used as markers?
(Note: any cut inside an antibiotic resistance site will destroy its ability to
produce antibiotic reSistance.)
Sources: The Science Teacher, April 1987.
Recombinant DNA and Biotechnology, ASM Press, 1996.
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