CHAPTER 3

BUILDING A RECOMBINANT PLASMID

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INTRODUCTION
In Chapters 1 and 2, you learned about four important tools of genetic
engineering: the micropipette, gel electrophoresis, plasmids, and restriction
enzymes. Chapter 2 focused on cutting DNA into fragments that could be
combined into a recombinant plasmid. In this chapter, you will ligate (paste
together) the fragments using a fifth tool that is needed to clone genes—DNA
ligase. DNA ligase is an enzyme that catalyzes (increases the rate of a reaction)
the joining of DNA fragments; it is one of several enzymes involved in DNA
replication in all cells. This same process is used to make recombinant plasmids
that contain the gene for human insulin, human growth hormone, blood
clotting factors, and other human therapeutic proteins.

CHAPTER 3 GOALS
By the end of this chapter, you will be able to do the following:
• Describe the role of a DNA ligase in replication
• Explain how DNA ligase is used to create a recombinant plasmid
• Describe possible recombinant plasmids that form when ligating a
restriction digest

WHAT DO YOU ALREADY KNOW?

Discuss the following questions with your partner and write your ideas in your
notebook. Be prepared to discuss your responses with the class. Don’t worry
if you don’t know all the answers. Discussing these questions will help you
think about what you already know about enzymes, DNA replication, and DNA
ligation (the reaction that chemically joins two fragments of DNA, resulting in a
recombinant DNA molecule).

1. What is the function of enzymes in reactions?

2. How does DNA replication occur?
3. Why is replication of DNA essential in all cells?
4. What happens when two DNA fragments with complementary sticky ends
join? How does the activity of DNA ligase ensure that the join is permanent?

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© 1991, 2000, 2013–2019 Amgen Foundation, Inc. All rights reserved.
 PASTING DNA FRAGMENTS TOGETHER
The discoveries of restriction enzymes, plasmids, and ligases in cells were
important in the quest to understand how bacteria grow and reproduce. But
it was understanding how these biomolecules could be applied to manipulate
DNA that launched a new era of genetic engineering, one in which humans
could directly engineer organisms at the genetic level and use them to produce
therapeutic proteins. Plasmids provided the vehicle to clone genes, and
restriction enzymes provided the means to generate the fragments of DNA
needed to form recombinant plasmids. The final essential ingredient was a way
to “glue” the fragments together.

DNA LIGASES IN NATURE

To better understand ligases and their role in cloning a gene, it is important to
understand the role they play in nature. In the early 1960s, scientists isolated
enzymes that had the ability to combine DNA fragments. These enzymes, called
DNA ligases, were shown to be involved in DNA replication of chromosomes and
plasmids. Replication occurs in a number of steps (as shown in Figure 3.1):

Figure 3.1: Summary of DNA replication

3. A new daughter
strand is formed
2. DNA polymerase continuously on
adds complementary this parent strand
DNA polymerase
nucleotides to each 3’
parent strand 5’
1. Two parent DNA
strands separate
at an ori site

DNA molecule

4. A second daughter strand

is formed discontinuously
as DNA fragments on this
5’ Fragment parent strand
3’
being made 3’
5’
DNA polymerase

5. Ligase catalyzes the DNA ligase

joining of the DNA
fragments

1. The two original strands begin to separate at multiple locations on the DNA
molecule. Each location is an origin of replication, or ori site (the same as
you learned about with plasmids in Chapter 2). The two original strands are
called parent strands.

2. Once the parent strands are separated and the bases are exposed, an
enzyme called DNA polymerase adds new nucleotides. Each new nucleotide
base forms hydrogen bonds with existing nucleotide bases on the parent
strand. Complementary base pairs are created in this step.

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3. On one parent strand, DNA polymerase adds nucleotides continuously to
form a new complementary strand, a daughter strand.

4. On the other parent strand, DNA polymerase adds nucleotides

discontinuously, resulting in small fragments of DNA. (To learn about why
one strand is replicated continuously and one discontinuously, refer to Did
You Know? DNA Directionality on page 57.)

5. The DNA fragments must be joined by the enzyme DNA ligase in order
to form the second daughter strand. The ligase joins the fragments by
catalyzing the formation of a covalent bond between adjacent nucleotides.

Once the replication steps are complete, two new DNA molecules, each
consisting of one parent strand and one daughter strand, have been made. Both
molecules are exact copies, or replicas, of the original DNA molecule.

CONSIDER:

• What are the roles of hydrogen bonds and covalent bonds in the structure
of DNA?

• Based on what you just learned about DNA replication, what is the possible
role of ligases in joining the sticky ends of DNA fragments?

ROLE OF LIGASES IN GENE CLONING

The role of ligase in gene cloning is similar to its role in replication in that it
binds DNA fragments together. In gene cloning, recombinant DNA is the result
of ligation of fragments from a restriction digest (as shown in Figure 3.2). Any
two fragments that have ends cut with the same restriction enzyme can be
ligated together. First, the unpaired nitrogen bases at the sticky ends form
hydrogen bonds to each other, and then the ligase catalyzes the formation of
covalent bonds between adjacent nucleotides. If you have multiple fragments,
the ligation procedure can lead to a number of possible products (see Figure
3.3).

Figure 3.2: Ligation of DNA fragments in genetic cloning

2. Ligase catalyzes the joining

1. Complementary of the DNA fragments
nucleotides in the together
sticky ends pair up
5’
G A Plasmid DNA
G G A 3’
3’ T C C C T A

C T 5’
Gene of interest C C T A G G G A T

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© 1991, 2000, 2013–2019 Amgen Foundation, Inc. All rights reserved.
 Figure 3.3: Process of Ligation

Starting Plasmids

pARA pKAN-R
4,872 bp 5,512 bp

Cut both with the same

restriction enzymes

Mix the products; they join

by base pairing to create
recombinants

DNA ligase creates

permanent bonds

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DID YOU KNOW?
DNA Directionality

DNA is a double helix of two intertwined strands that are hydrogen-

bonded together through the bases of the nucleotides. Each strand
has a different directionality, meaning that each strand has a chemical
orientation based on the chemical group that it ends with. The 5’ (5
“prime”) end of the DNA strand is the end that has the fifth carbon in
the deoxyribose sugar ring. The 3’ (3 “prime”) end of the DNA strand
is the end that has the hydroxyl (OH) group on the third carbon in the
deoxyribose sugar ring (see Figure 3.4).

This naming of the ends

Figure 3.4: DNA structure of the strands is useful in
understanding how DNA
5’ end
A T OH
polymerase works. DNA
3’ end
polymerase can only assemble
new DNA strands in the 5’
A T
to 3’ direction, adding new
nucleotides to the 3’-hydroxyl
G C group. Therefore, on one
strand replication occurs

C G continuously as polymerase
adds nucleotides in the 5’ to 3’
direction to form the daughter
T A strand. On the other strand,
the DNA polymerase also adds

T A nucleotides in the 5’ to 3’
3’ end direction, which results in short
OH
5’ end fragments of DNA that must be
ligated together to form the
other daughter strand.

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© 1991, 2000, 2013–2019 Amgen Foundation, Inc. All rights reserved.
LABORATORY

LABORATORY 3:
BUILDING THE pARA-R PLASMID
In this laboratory you will ligate the DNA fragments you produced during
Laboratory 2, using DNA ligase to make new recombinant plasmids. These
recombinant plasmids will contain the four restriction fragments (the pieces of
DNA that result from cutting the DNA molecule with a restriction enzyme) from
Laboratory 2, recombined in different ways to produce new sets of DNA. The
ligation process will result in several different plasmids, but the plasmid that you
are interested in will contain the gene for ampicillin resistance (ampR), the red
fluorescent protein (rfp) gene, a promoter for initiating transcription (pBAD),
the arabinose activator sequence (araC), and the ori sequence for the initiation
of DNA replication. This desired recombinant DNA plasmid is called the pARA-R
plasmid (see Figure 3.5).

Figure 3.5: The pARA-R plasmid

BamHI
pBAD

pBAD-rfp
Hin
IId

During the lab, you will mix together the DNA fragments from your restriction
digest in Laboratory 2 and the DNA ligase, but you will not be able to observe
anything until Laboratory 4, when you will have the opportunity to separate and
identify your DNA molecules using gel electrophoresis. However, you will prepare
for what you might observe by determining the possible plasmids that can occur
and then drawing diagrams of these plasmids. In this work, you are modeling the
process of ligation.

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 LABORATORY

BEFORE THE LAB

Discuss the following with your group, record your thoughts, and be prepared to
share your responses with the class.

1. Review your answer to question 1 in Before the Lab for Laboratory 2 (page
44), in which you described the fragments that formed from the digestion
of pKAN-R and pARA with BamHI and HindIII. Using this information, draw
three possible recombinant plasmids resulting from the joining of two pARA
and pKAN-R fragments. For each plasmid, identify the genes, other important
sequences, and the number of base pairs each has.

2. Read through the Methods section on page 60 and briefly outline the steps,
using words and a flowchart.

MATERIALS
Reagents

• A rack with the following:

 Microfuge tube of digested pKAN-R from Laboratory 2 (K+)
 Microfuge tube of digested pARA from Laboratory 2 (A+)
 Microfuge tube of 5x ligation buffer (5xB)
 Microfuge tube of DNA ligase (LIG)
 Microfuge tube of distilled water (dH2O)

Equipment and Supplies

• 80°C water bath with floating microfuge tube rack (will be shared among
all groups)
• P-20 micropipette
• Tip box of disposable pipette tips
• Permanent marker
• Microcentrifuge (will be shared among all groups)
• Waste container for used tips and microfuge tubes (may be shared
with another group)

SAFETY:

• All appropriate safety precautions and attire required for a science

laboratory should be used, including safety goggles. Please refer to your
teacher’s instructions.

• Wash your hands well with soap after completing the lab.

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LABORATORY

METHODS
1. Check your rack to make sure that you have all the reagents listed.
2. Place K+ and A+ tubes from Laboratory 2 in the 80°C water bath (you will
place your tubes in the floating microfuge tube rack; once each group’s
samples are loaded, your teacher will place the rack in the water bath) for
20 minutes. This heat exposure will denature (inactivate) the restriction
enzymes.

NOTE: During the incubation, share and discuss your answers to question
1 in Before the Lab. Also share and discuss your answer to the STOP AND
THINK question that follows, and begin answering the Chapter 3 Questions
on page 61.

STOP AND THINK: Why is it important to inactivate the BamHI and HindIII
restriction enzymes before ligating the fragments? What might happen if
you did not perform this step?

3. Label the LIG tube with your group number and class period.
4. After 20 minutes, remove the K+ and A+ tubes from the water bath and
place them in your rack.
5. Using a new pipette tip with each reagent, add the following solutions
directly into the solution at the bottom of the LIG tube:
a. 4.0 μL of A+
b. 4.0 μL of K+
c. 3.0 μL of 5xB
d. 2.0 μL of dH2O

LAB TECHNIQUE: In steps 5a–d, be sure to use a new micropipette tip

for each reagent to avoid contamination.

6. After adding the dH2O, gently pump the solution in and out with the pipette
to mix the reagents. Cap the tube when done.
7. Spin the LIG tube in the microcentrifuge for several seconds to pool the
reagents at the bottom of the tube.

LAB TECHNIQUE: Distribute the tubes evenly in the microcentrifuge so that

their weight is balanced.

8. Place your LIG, A+, and K+ tubes in the microfuge racks designated by your
teacher. (Your LIG tube will incubate at room temperature until the next
class. Your A+ and K+ tubes will be returned to the –20°C freezer for use in
Laboratory 4.)

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