Key Words (GENETIC ENGINEERING)

Genetic engineering: the process of using biotechnology to alter the genome of an organism,
typically with the goal of conferring some desirable trait.

Genetically Modified organism: an organism with genetic material that has been altered using
genetic engineering technology.

Transgenic organism: a genetically modified organism that contains foreign genetic material from a
different species.

Cisgenic organism: a genetically modified organism that contains foreign genetic material from the
same species.

Recombinant Plasmid: a circular DNA vector that is ligated to incorporate a gene of interest.

Insulin: a hormone secreted by the pancreas to control blood glucose levels.

Gene of interest: a gene scientists want to be expressed in recombinant bacteria.

Vector: a means of introducing foreign DNA into an organism.

Plasmid vector: a piece of circular DNA that is modified to be an ideal vector for bacterial
transformation experiments

Transgenic organism: a genetically modified organism that contains foreign genetic material from a
different species.

Gene sequencing: The process of determining the nucleotide sequence in an organism’s genome.

Genetic modification: the manipulation of an organism’s genetic material using biotechnology

Gene therapy: repairing genetic mutations by replacing a defective gene with a healthy one.

Gene knockout: a technique in gene editing where scientists prevent the expression of a target gene
to understand its function in an organism.

Transgene: a segment of DNA from one organism that is introduced into the genome of another
organism, typically from a separate species.

Host organism: the organism which researchers wish to genetically modify, typicaly via the
alteration of its genome and/or the insertion of a genome.

RECOMBINATION AND TRANSFORMATION

Explain how plasmids can be used to make bacteria produce human insulin?

The gene for insulin is cut from human DNA and inserted into plasmids that have been cut with the
same restriction enzyme, and the sugar-phosphate backbone is repaired by DNA ligase. The
recombinant plasmids include the ampR gene (antibiotic resistance) and lacZ (reporter gene which
produces B-galactosidase that converts X-gal from colourless to blue compound). The recombinant
plasmids are then introduced to bacteria via a method such as heat shock therapy or
electroporation. Only some bacteria will take up the recombinant plasmids, and these are selected
for using antibiotics. The transformed bacteria are colourless due to the dysfunctional lacZ gene,
and are cultured, and then transcribed and translated to produce human insulin. The β-
galactosidase tails are removed and mixed together, which allows disulphide bonds to form and
create functional human insulin.

Explain the benefit of using enzymes that produce sticky ends instead of blunt ends.

The enzyme cuts the plasmid results in complementary sets of overhanging nucleotides, which
means that other fragments of DNA that have also been cut with the same enzyme can form
hydrogen bonds between the complementary base pairs of each strand. Additionally, the blunt ends
enzyme can result in target fragments being inserted back-to-front. Both of these factors mean that
sticky end enzymes makes successful recombination more likely than blunt end enzymes.

Describe the purpose of heat shocking.

Heat shocking bacteria aims to increase the uptake of plasmids by bacteria. The rapid change in
temperature is responsible for increasing the permeability of the bacteria’s plasma membrane,
which allows plasmid vectors to cross the phospholipid bilayer and enter the bacteria’s cytoplasm.

Describe the role of DNA fragments.

To join fragments of DNA together.

Explain what sticky end restriction enzymes are and how they can be useful.

Sticky end restriction endonucleases cleave DNA with overhanging additional nucleotides. These
overhanging sections are capable of joining with complementary fragments together in the correct
orientation.

What is meant by the term ‘transform’ when creating transformed bacteria?

‘Transform’ refers to the uptake of plasmids by bacteria.

How are restriction enzymes used in bacterial transformation?

Restriction endonucleases are used to cut the plasmid vector and the gene of interest, producing
complementary ‘sticky ends’ that allow the gene of interest to be inserted into the plasmid.

Describe how DNA ligase is used to help insert a gene coding for a human protein into this
plasmid.

DNA ligase joins the gene of interest and plasmid vector together by forming phosphodiester bonds
between each DNA sugar-phosphate backbone.

What characteristic of the genetic code enables a human protein to be made by bacterial cells?

The genetic code is universal, meaning that a human gene can be expressed by bacteria and its
downstream protein can be produced, despite the gene coming from a different species.

Non-pathogenic strain of bacteria.

The strain of bacteria is unable to cause disease and contains foreign DNA.
PRACTISE SAC
The genetic engineering tool CRISPR is mentioned in the article as “molecular scissors”. What is
another name for molecular scissors and how do they work?

Restriction endonucleases. Attach to their specific recognition site on the DNA, cleaving the
phosphodiester bond of the sugar-phosphate backbone that holds the DNA nucleotides together.

What might “off target effects” be and why may they be a problem?

Off target effects are where DNA is cut outside of the targeted gene. This may cause other non-
targeted genes to be affected or switched off.

Define the term gene therapy.

Gene therapy is the introduction of normal genes into DNA in cells in place of defective genes in
order to correct genetic disorders.

Outline the process where a gene can be inserted into cells using a virus.

Step 1: Cut normal gene from a healthy cell with restriction enzymes and the viral DNA with same
restriction enzyme creating matching sticky ends.

Step 2: Join normal gene to viral DNA with DNA ligase.

Step 3: Use the viral vector to carry the normal gene to the patient’s own blood cells

Step 4: The virus inserts the normal DNA into the cell where the DNA is incorporated into the cellular
DNA and is expressed

PROCESS OF BACTERIAL TRANSFORMATION

Step 1: The gene of interest is generated and an appropriate plasmid vector is chosen.

Step 2: Same restriction enzymes are used to create complementary sticky ends on both the gene of
interest and the plasmid vector.

Step 3: DNA ligase is added to join the gene of interest and plasmid vector by sealing the sugar-
phosphate backbone.

Step 4: Recombinant plasmids and bacteria are mixed together in a solution. Electroporation or heat
shock increases the bacteria’s membrane permeability, allowing plasmids to pass into the cytoplasm.

Step 5: Bacteria are cultured on an antibiotic-containing agar plate. Only transformed bacteria are
able to grow and form colonies.

What is a vector? Provide one other example of a vector that may be used in genetically modifying
organisms.

A vector in this context is used as a vehicle to transport foreign genetic material into another cell
where it can be replicated or expressed. Bacterial plasmids may also be used as vectors.

Explain why editing a gene is easier to do outside of the body and why editing a gene inside the
body while it is more difficult is preferable.

Gene editing is easier outside the body as the affected cells can be isolated outside of the body and
individual cells treated (1) and tested to ensure the cell expresses a gene before placement (1). It is
preferable to gene edit inside the body as more cells can be modified (1) and as the cells are in situ
any cells modified will be immediately effective.

Each of the life changing breakthroughs that are provided in the articles have pros and cons. List
and explain two biological, two social and two ethical considerations raised in the articles.

Biological

Vectors such as viruses can infect more than one type of cell and so may change the genetic material
in non-target cells.

ii. Target cells may not take up the vector making the therapy less effective

iii. The DNA may be inserted into the wrong location adversely affecting gene expression

iv. The transferred genes may be overexpressed causing excessive protein production which may
adversely affect the patient

v. The vector may initiate an immune response causing the vector to be removed

Social

The cost associated with treatment can be exorbitant and not everyone can afford it

ii. Techniques are experimental and be playing God

iii. Could unknown side effects be treated?

iv. Who owns the rights to a genetic treatment?

Ethical

i. Safety – The possibility of off target effects (editing the wrong place) or some cells carry the
edit while others do not may lead to medical issues arising as an unintended effect.
ii. ii. Use for non-therapeutic purposes may cause medical issues and possible unknown side
effects
iii. iii. Lack of informed consent if used on germline cells or embryos/babies iv. Justice and
equity concerns with expensive therapies only being available to the wealthy with
disparities in health care
iv. v. The use of embryos is controversial on moral and religious grounds

Explain why a person with Hunters disease may be missing an enzyme and how cutting DNA in the
right spot can produce that enzyme

A person with Hunters gene must have had a (missense or nonsense) mutation in their DNA (1)
which when expressed made the enzyme not functional (missense) or not transcribed at all
(nonsense) (1). By cutting at the site of the mutation the DNA can be replaced with the correct DNA
code for the enzyme (1) which can then be successfully expressed.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

Key Words
CRISPR-CAS9: a complex formed between gRNA and CAS9 which can cut a target sequence of DNA.
Used for protection from viruses.

Endonuclease: an enzyme that breaks the phosphodiester bond between two nucleotides in a
polynucleotide chain.

CRISPR: short, clustered repeats of DNA founds in prokaryotes which protect them against viral
invasion.

Spacer: short sequences of DNA obtained from invading bacteriophages that are added into the
CRISPR sequence.

Protospacer: a short sequence of DNA extracted from a bacteriophage by Cas1 and Cas2, which has
yet to be incorporated into the CRISPR gene.

Protospacer adjacent motif: a sequence of DNA nucleotides that is found immediately next to the
target DNA by Cas9.

Guide RNA (gRNA): RNA which has a specific sequence determined by CRISPR to guide Cas9 to a
specific site.

What is CRISPR?

CRISPR is a naturally occurring sequence of DNA found in bacteria that plays an important role in
their defence against viral attacks.

What action is Cas9 expected to perform?

Cas9 is expected to cut the DNA at a specific sequence, complementary to the gRNA.

What are the applications of CRISPR in eukaryotes?

Research, agriculture, genetic diseases.

Identify the components that make up a monomer of gRNA.

Ribose sugar, phosphate and nitrogenous base.

CRISPR-Cas9 is naturally occurring in prokaryotic organisms? What is the purpose of this

machinery in prokaryotes?

CRISPR-Cas 9 cuts target DNA, according to the complementary sequence provided by gRNA, to
combat bacteriophage invasion.

What is meant by the term restriction endonuclease?

An endonuclease that cuts DNA at a specific restriction site.

Explain the role of restriction endonuclease.

Restriction endonucleases cuts DNA at a specific recognition site.

Suggest an advantage of using Cas9 instead of a restriction endonuclease.

Cas9 is versatile in that it can be programmed to target any specific sequence dictated by a piece of
gRNA, whereas restriction endonucleases will only act on their specific restriction site, which cannot
be programmed.

Introns are spliced out during post-transcriptional modifications. How can this splicing event give
rise to different functional proteins?

Through alternative splicing, particular exons can be included or excluded from a final gene product.
This can alter the translated sequence resulting in different functional proteins.

Describe one example of how CRISPR-Cas9 could be used in understanding the function of introns.

Through gene knockout, an intron could be disrupted and any changes can be observed to
determine its function.

What happens when the viral DNA is cut?

When the viral DNA is cut, enzymes within the bacterium will naturally act to repair it. However the
repair mechanisms in a cell are prone to errors that can result in nucleotide additions, deletions and
insertions in the middle of the viral gene. These mutations can render the viral gene non-functional.
If mutation does not occur, gRNA will find the gene again and repeat the whole process until the
DNA repair mechanisms induce a mutation, inactivating the virus.

How to use CRISPR-Cas9 for gene editing.

1. Synthetic gRNA is created in a lab that has complementary spacer to the target DNA that
scientists wish to cut.
2. A Cas9 enzyme is obtain with the appropriate target PAM sequence.
3. Cas9 and gRNA are added together in a mixture and bind together to create the CRISPR-Cas9
complex
4. The gRNA-Cas9 mixture is then injected into a specific cell, such as a zygote.
5. The Cas9 finds the target PAM sequence and checks whether the gRNA aligns with the DNA.
6. Cas9 cuts the selected sequence of DNA
7. The DNA has a blunt end cut that the cell will attempt to repair
8. When repairing the DNA, the cell may introduce new nucleotides into the DNA at this site.
Scientists may inject particular nucleotide sequences into the cell with the hope that it will ligate
into the gap.

How CRISPR-Cas9 defence system works.

1. Exposure- bacteriophage injects its DNA into a bacterium, which identifies the viral DNA as a
foreign substance. Cas 1 and Cas 2 enzymes cut out a short section of the viral DNA known as a
protospacer. This protospacer can then be introduced into the bacterium’s CRISPR gene and
become a spacer.
2. Expression- the CRISPR spaces are transcribed along with half a palindrome from the repeat
either side of it, and converted into a RNA molecule known as guide RNA (gRNA). gRNA binds to
Cas9 to create CRISPR-Cas9 complex which Is directed to the viral DNA inside the cell that is
complementary to the gRNA.
3. Extermination- the CRISPR-Cas9 complex then scans the cell for invading bacteriophage DNA
that is complementary to the ‘mugshot’ on the gRNA. When it does, Cas9 cleaves the
phosphate-sugar backbone to inactivate the virus. Cas9 contains two active sites to cut both
strands of DNA and create blunt ends.
What role does DNA ligase play?

DNA ligase joins the DNA that has been cut by the Cas9 enzyme.

Polymerase Chain Reaction

Primer: a short, single strand of nucleic acids that acts as a starting point for polymerase enzymes to
attach.

Outline the steps in the polymerase chain reaction.

In the denaturing stage, the mixture is heated to approximately 90-95 C to break the hydrogen
bonds and denature the double-stranded DNA, separating the strands in the sample. In the
annealing stage, the DNA sample is cooled to approximately 50-55 C to allow the primers to bind to
complementary sequences on the single-stranded DNA. In the elongation stage, Taq polymerase
copies the strands by adding complementary nucleotides, extending the primers. The cycle is then
repeated to produce more copies of the target DNA sequence.

Name the process to amplify DNA.

Polymerase chain reaction.

Explain the role of the polymerase enzymes.

The polymerase enzyme catalyses the production of a new strand of DNA by extending the primers.

Explain the purpose of the polymerase chain reaction.

The polymerase chain reaction amplifies a sample of DNA to increase the quantity of DNA available.

Explain the role of primers.

DNA primers are added to the mixture to bind to complementary nucleotide sequences at 5’ ends of
each single-stranded piece of DNA. This provides Taq polymerase with a binding site to begin
building a complementary strand.

Two primers are needed as the 5’ end of the coding and template strand are different.

Justify why Taq polymerase must be used instead of human DNA polymerase.

Taq polymerase has a very high optimal temperature, working optimally at 72 C, whereas human
DNA polymerase would denature and be incapable of synthesizing a new strand at this
temperature.

Gel Electrophoresis
Gel electrophoresis: a technique that separates DNA fragments based on their molecular
size.
Well: an indent in the gel into which a DNA sample is loaded
Standard ladder: a mixture of DNA fragments of known length that is used to determine
the size of fragments in a sample.
Agarose gel: a sponge-like gel used in electrophoresis that contains pores for DNA
fragments to move through.
Buffer: an ion-rich solution that carries electric current through the agarose gel.
Genetic testing: screening an individual’s DNA for any abnormalities that may make them
susceptible to a particular disease or disorder.
DNA profiling: the process of identification on the basis of an individual’s genetic
information
Name two properties of DNA fragments that allow them to be separated from each other
during gel electrophoresis.
Differing molecular size and a partial negative charge.
Other than factors relating to the DNA sample, identify one factor that will impact the
rate of movement of DNA fragments through the agarose gel.
Voltage or power applied to the gel.
Why is there only one band in lanes 2 and 4 but two bands in lanes 3, 5 and 6.
The individuals corresponding to lanes 2 and 4 are homozygous for that particular gene,
and so contain twice the amount of DNA for that allele as all their fragments are of equal
size, resulting in a thicker band. The lanes with two bands contain DNA samples from
heterozygous individuals and so contain fragments of two different sizes.
What is the purpose of a standard ladder?
A standard ladder allows for the measurement of fragment length by comparing the
distance it has moved through the gel to a series of fragments with a known size.
Outline two safety guidelines.
The electrophoresis equipment is turned off to avoid electrocution and wear safety glasses
to protect the eyes.
Why might an ethics board advise this experiment has a small sample size?
To reduce the number of embryos destroyed/
Explain why the embryos should not be implanted into a surrogate mother after this
experiment?
As the technology is still being studied, it would be unethical to birth the embryo as there
could be unforeseen harmful effects to the mother or the embryo.
Why experiment ex vivo not in vivo in adults.
The experiment was completed ex vivo instead of in vivo to create a controlled
environment and prevent the influence of other factors on the experiment and prevent
potentially harmful side affects on the subject. The experiment was performed on single-
celled embryos so the entire organism has the altered gene, instead of single cells, which
would be the case if it was performed in adults.
 CHAPTER 2: PROTEINS AND NUCLEIC ACIDS
Describe how the functional 3D structure of a protein is formed.
The functional 3D structure of a protein is formed by the interactions between the R-groups
of amino acids within the polypeptide chain.
Suggest how the functional diversity of proteins arises.
The functional diversity of proteins arises through the ability to create many combinations
of amino acids and polypeptide chains of differing length, thereby allowing proteins to fold
into different functional structures.
Indentify two other functional roles of proteins in living organisms.
Proteins transport substances across membranes and defend against pathogens.
What information is obtained from protein sequencing?
The primary structure of a protein, which is its sequence of amino acids.
What information is obtained from gene sequencing?
The sequence of nucleotides in a gene.
Degenerate: many different codons can code for the same amino acid.
Unambiguous: each codon is only capable of coding for a single amino acid
Non-overlapping: each triplet or codon is read independently of adjacent triplets or
codons.
Universal: nearly all organisms use the same set of rules and codons to code for proteins.

Promoter: binding site of RNA polymerase, denoting the starting position of transcription.
Termination Sequence: a sequence of DNA which signals for the end of transcription.
Operator: binding site for repressor proteins
Introns: non-coding regions of DNA
Exons: coding regions of DNA.
Identify three key differences in structure between mRNA and DNA.
mRNA is single-stranded while DNA is double-stranded. mRNA contains a ribose sugar, while
DNA contains a deoxyribose sugar. mRNA contains the nucleotide base uracil, while DNA
contains thymine. DNA has a double helix while mRNA has a linear shape.
While mutations can often lead to the production of polypeptides with different amino
acid sequences, sometimes mutations still produce the same amino acid. Explain how this
may occur.
Mutations may not lead to the production of a different amino acid due to the degenerate
nature of the genetic code, which allows for multiple different codons or triplets to code for
the same amino acid.
Describe the difference between introns and exons.
While introns are regions of non-coding DNA, exons are regions of coding DNA.
Describe the purpose of the promoter region of a gene.
The promoter regions serves as the binding site for RNA polymerase, which denotes the
starting position of transcription.
Describe the role of anticodons in translation.
The tRNA anticodon is complementary to the mRNA codon being read by the ribosome. The
anticodon attaches to the mRNA codon, which then allows for the corresponding amino acid
carried by tRNA to be joined to the growing polypeptide chain.
TRANSCRIPTION
Transcription involves the unwinding of the DNA helix which allows for the binding of RNA
polymerase to the promoter region. RNA polymerase then synthesizes a strand of pre-
mRNA with the use of complementary RNA bases. In RNA, the base thymine is replaced by
uracil. Transcription is terminated when RNA polymerase reaches termination sequence and
the pre-mRNA strand is released and undergoes post-transcriptional modifications.
TRANSLATION
Translation begins with the binding of an mRNA molecule to a ribosome, with the start
codon initiating the process. tRNA molecules with anticodons complementary to the mRNA
codons transport specific amino acids to the ribosome, which are added to the growing
polypeptide chain via peptide bonds. Translation terminates once the stop codon is
recognized and the polypeptide chain is released.
RNA PROCESSING
A methyl-G cap is added at the 5’ end and a poly-A tail is added at the 3’ end. The removal
of introns and the splicing of exons together.
Explain the purpose of the methylated cap and poly A-tail on mRNA.
The cap and tail are required for orientation to ensure the mRNA is read in the correct
direction. Serve to stabilize the mRNA molecule, preventing it from degrading and allowing
it to bind to ribosomes during translation.
Describe Transcription.
Involves the production of pre-mRNA from the nucleotide code of the DNA template strand.
Describe Translation.
Involves the interpretation of the mRNA strand at a ribosome to form a polypeptide chain.
Outline RNA Processing.
Introns are removed from the pre-mRNA strand and exons are spliced together to form an
mRNA strand containing only coding segments. A poly-A-tail is added to the 3’ end a methyl-
G-cap is added to the 5’ end of the mRNA molecule.
Describe the role of the two types of genes.
Structural genes are responsible for producing proteins that form the structure or facilitate
the functioning of an organism. Regulatory genes are responsible for producing proteins
that influence the expression of other genes.
Suggest one safety consideration.
Practising appropriate hand hygiene.
Suggest why an organism regulates the expression of a gene.
Regulating the expression of a gene saves energy, because the gene product is only
produced when it is needed.
What is meant by the term gene regulation.
Gene regulation describes any mechanism that acts to increase or decrease the production
of specific gene products in a cell.
Explain how high levels of tryptophan in a cell lead to the regulation of the tryptophan
gene.
Tryptophan binds to a repressor protein which then has a conformational change. In its
changed state, the repressor protein binds to the operator, downstream of the promoter
region but upstream of the operon, blocking RNA polymerase from transcribing the operon.
Operon: a cluster of linked genes (structural genes in prokaryotes) that all share a common
promoter and operator and are transcribed at the same time.
Outline the secretory pathway.
The proteins, which are synthesized at ribosomes attached to the rough endoplasmic
reticulum, are folded within the rough endoplasmic reticulum and transported to the Golgi
apparatus in transport vesicles. At the Golgi apparatus, the proteins are modified before
being packaged into secretory vesicles. The secretory vesicle fuses with the plasma
membrane, releasing the proteins into the extracellular environment via exocytosis.