Name: _______________________________________
Date: ____________________________ Hour: _______
Lab:
Recombinant Paper Plasmids:
Background Reading:
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. You
could make a recombinant bicycle by disassembling two bicycle by disassembling two bicycles and
reassembling them in a new way: putting the wheels of one on the frame of the other, for example. 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. Often, one of these DNA sources is a plasmid.
Plasmids are small, circular DNA molecules that can reside in cells. Plasmids are copied by the cell’s DNA
replication enzymes 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. As you might expect, bacterial and other chromosomes have replication origin, it cannot be
copied by the cell and will not be transmitted to future generations.
Plasmids often contain genes for resistance to antibiotics. Antibiotics are natural substances produced mostly
by soil microorganisms. Antibiotic production allows these microorganisms to kill off competing microbes.
Antibiotic resistance is also a natural phenomenon; at the very least, the antibiotic producers must be resistant to
the antibiotics they make! We will be working with genes fro resistance to the antibiotic ampicillin and
kanamycin.
In this activity, we will assemble plasmids carrying genes for ampicillin and kanamycin resistance and then
recombine the two plasmids. We call the plasmid with ampicillin resistance pAMP, the plasmid with
kanamycin resistance pKAN, and the recombinant plasmid pAMP/KAN. We will use paper plasmid DNA
models to go through the process that scientists use when making recombinant DNA. Scissors will substitute
for restriction enzymes. The enzyme DNA ligase, which forms phosphodiester bonds between pieces of DNA,
is represented by scotch tape. Our result will be a model of a recombinant DNA molecule. Scientists place real
recombinant plasmids back into bacteria where they multiply. The bacteria also multiply, making millions of
copies of the recombinant DNA molecule and the proteins it encodes.
Construction of the pAMP and pKAN Plasmids
Locate the three strips of DNA code on the worksheet (Appendix A) marked “Paper pAMP model.” On each
strip, the two rows of letters indicate the nucleotide bases, and the solid horizontal lines indicate the sugarphosphate backbone of the DNA molecule. The hydrogen bonds between the base pairs are located in the white
space between the rows of letters.
1. Use your scissors to cut carefully along the solid lines. Cut out each strip, leaving the solid lines intact.
Make a vertical cut to connect the open end of the box formed by the solid lines. This cut will remove
the 5’ and 3’ from the strip.
2. After all three strips are cut out, glue or tape the “1” end to the “paste 1” area, covering the vertical lines.
Connect the “2” to “paste 2” and “3” to “paste 3” until you complete the circular model of the pAMP
plasmid.
3. Using the page (Appendix A) marked “Paper pKAN plasmid model,” cut out and paste together a
plasmid containing a kanamycin resistance gene. The procedure is exactly the same as for the pAMP
plasmid.
Constructing a Recombinant pAMP/pKAN plasmid
You have now prepared a pAMP plasmid and a pKAN plasmid. In this pare of the activity, you will use them
as starting materials to make a recombinant plasmid. You will cut pAMP and pKAN with two specific
enzymes, BamHI and HindIII. You will ligate together fragments that come from each plasmid, creating a
pAMP/KAN plasmid.
1. First, simulate the activity of the restriction enzyme BamHI. Reading from 5’ to 3’ (left to right) along
the top row of your pAMP plasmid, find the base sequence GGATCC. This is the BamHI restriction
site. Notice that it is a palindrome. Cut through the sugar-phosphate backbone between the two G’s,
stopping at the center of the white area containing the hydrogen bonds (don’t cut all the way through the
paper). Do the same on the opposite strand. Cut through the hydrogen bonds between the two cut sites,
and open the plasmid into a strip. Each end of the strip should have a single-stranded protrusion with
the sequence 3’ CTAG 5’. Mark the ends of the strip BamHI.
2. Next, simulate the activity of the restriction enzyme HindIII. Reading from 5’ to 3’ (left to right) along
the top row of the pAMP strip, find the sequence AAGCTT. This is the HindIII restriction site. It is
also a palindrome. Cut the sugar-phosphate backbone between the two A’s, stopping at the center of the
white space containing the hydrogen bonds. Repeat the cut between the two A’s on the opposite strand
of the restriction site. Cut the intervening hydrogen bonds. This time you should have two pieces with
single-stranded protrusions. One protrusion on each piece is the BamHI end; the other should have the
sequence 3’ TCGA 5’. Mark each of these two new ends HindIII. You now have two pieces of pAMP
DNA. Set them aside.
3. Using your pKAN plasmid model, repeat steps 1 and 2. The pKAN plasmid is now in two pieces with
labeled ends. Including the two pieces of pAMP, you now have four pieces of plasmid.
4. Take the piece of plasmid with the ampicilllin resistance gene in it, and connect it to the piece
containing the kanamycin resistance gene by using DNA ligase (scotch tape). Be sure that
complementary bases are p aired where you make the ligations. Notice that the BamHI end will not pair
with the HindIII end but will pair with another BamHI end. Likewise, the HindIII end must pair with
another HindIII end.
Remember that DNA is three dimensional. In our model, the letters representing the bases can look
upside down, but in real DNA, it doesn’t matter. O in this paper simulation, the letters representing the
bases do not need to be right side up. All that matters is that the 5’ – to – 3’ directions match within a
strand and that the base pairs are correct.
You have now created a recombinant pAMP/pKAN plasmid.
Transformation:
It is possible to introduce plasmids into bacterial cells through the process of transformation. Bacteria that can
be transformed (can take up DNA) are called competent. Some bacteria are naturally competent. Others can be
made competent by chemical and physical treatment. After the bacteria absorb the plasmid DNA, they express
the new antibiotic resistance genes as instructed by the new DNA. Bacteria that express new proteins in this
way are said to be transformed. New copies of the plasmid are synthesized by the cell’s DNA replication
enzymes and passed to daughter ells as the bacteria multiply. Because many identical copies of the new genes
are generated in this process, you are said to have cloned the genes.
After transformation by plasmids containing antibiotic resistance genes, transformed bacteria can be detected by
plating on media containing the antibiotic(s). Only bacteria expressing the new antibiotic resistance genes
(transformed bacteria) can form colonies on the antibiotic plates. This method of selecting transformants
(because they are the only ones that can grow on the media) is a big advantage, because transformation is
usually very inefficient. In a typical experiment, less than 1 cell in 1, 000 will became transformed.
Classroom Activities: Manipulation of Analysis of DNA
Analysis:
1. Diagram the paper plasmids before you recombine them. Use B = BamHI, H = HindIII., ori = origin,
Ap = Ampicillin and Km = Kanamycin
2. The DNA scissors= __________________________________________(what was used in this lab)
3. The DNA Tape = ____________________________________________ (what was used in this lab)
4. How do scientists identify and isolate the new recombinant plasmids. Hint: see the paragraph on
transformation