unit 3 aos 1

EUKARYOTIC PROTEIN SYNTHESIS

What are the steps of bacterial transformation?

💛 Amplify gene by PCR →

💚 Create recombinant plasmid (with antibiotic resistance and reporter gene)

💜 Make bacteria competent with calcium chloride →

❤️ Use heat shock to transform bacteria →

🩵 Select successful transformants using antibiotics and reporter genes →

💙 Extract protein product for use.

What is a plasmid?

A small, circular, extrachromosomal DNA molecule naturally occurring in bacteria.

What do plasmids typically contain?

Antibiotic resistance genes, promoters, multiple restriction enzyme sites, and sometimes operons (if multiple genes must be expressed together).

What do antibiotic resistance genes on plasmids allow?

They allow selection of transformed bacteria by killing non-transformants with antibiotics.

Why is the small size of plasmids significant?

It makes them easier to isolate, cut with restriction enzymes, and reinsert into bacteria. Also, DNA is universal, so plasmids can express eukaryotic genes in bacteria.

Step 1 of bacterial transformation (gene amplification)

Amplify the gene of interest using PCR. Primers are designed to attach to the 5’ ends of the target gene.

Step 2 of bacterial transformation (creating recombinant plasmid)

Cut both the gene of interest and plasmid with the same restriction enzyme. Sticky ends anneal, and DNA ligase seals the sugar-phosphate backbone.

What occurs in bacterial transformation (steps 3 and 4)?

Competent bacteria uptake the recombinant plasmid via transformation (usually with calcium chloride and heat shock), then express and replicate the gene product via binary fission.

What does transformation mean?

Uptake of foreign DNA (often a plasmid) by a bacterial cell, enabling it to express the genes on that DNA.

Why do sticky ends improve the transformation process?

They improve efficiency by allowing complementary base pairing between the plasmid and gene insert.

How are competent bacteria created for (step 3 in bacterial) transformation?

calcium chloride binds to the membrane, which become positively charged (+ion).

- plasmids are negatively charged (because of DNA's backbone) so they're attracted to the positive charge and move and stick to the membrane of the bacteria

How are successful transformants selected and screened?

Antibiotics are added to agar plates. Only bacteria that took up plasmids with antibiotic resistance survive. Reporter genes can also be used to confirm gene expression.

Does having the plasmid mean the gene is expressed?

Not necessarily. A reporter gene is used to confirm gene expression by producing a visible change (like colour or fluorescence).

What do white colonies indicate in blue-white screening?

The gene of interest has interrupted the beta-galactosidase gene — indicating successful insertion and expression.

What do blue colonies indicate in blue-white screening?

The gene of interest is placed next to the beta-galactosidase gene as a fusion protein — indicating the gene was not inserted or not interrupting the original function.

Why are many medicines created recombinantly?

Recombinant DNA technology allows large-scale, fast, and ethical production of important proteins like insulin by inserting human genes into bacteria or yeast, which then produce the protein.

What is a reporter gene and what is it used for?

A gene used in genetic engineering to visually indicate whether a gene has been successfully inserted or expressed — often by producing a colour change or fluorescence.

What is the purpose of blue-white screening?

To check whether the correct protein is expressed by distinguishing recombinant from non-recombinant colonies.

What is the beta-galactosidase gene used for?

It produces the enzyme beta-galactosidase, which enables colour-based screening of transformed colonies.

What does the beta-galactosidase enzyme do?

It converts the substrate X-gal (in the presence of IPTG) into a blue dye, turning colonies blue when functional.

What do blue colonies indicate in blue-white screening?

The gene of interest was not inserted — the beta-galactosidase gene is still functional.

How are the alpha and beta chains of insulin produced recombinantly and why?

They are produced separately in different plasmids (bacteria) because insulin is made of two chains. The chains are later purified and joined to make functional insulin.

What is the first step in creating recombinant insulin and why are introns removed?

The insulin alpha and beta genes are created using PCR or extracted from a cell. Introns are removed because bacteria cannot process introns.

How is the insulin alpha gene prepared for insertion into a plasmid?

The insulin alpha gene is cut using a restriction enzyme to generate sticky ends for ligation.

What is done to the plasmid after inserting the insulin gene?

The plasmid (e.g. Plasmid A) is cut using the same restriction enzyme to produce matching sticky ends and contains an antibiotic resistance gene and a beta-galactosidase gene.

How is the recombinant plasmid formed?

The insulin alpha gene and plasmid are combined. Their sticky ends anneal, and DNA ligase seals the sugar-phosphate backbone, forming a recombinant plasmid.

How is recombinant plasmid introduced into bacteria?

Using the heat shock method (transformation) to insert it into Bacteria A.

How are transformed bacteria selected?

Using antibiotic resistance and blue-white screening to identify bacteria that successfully contain the recombinant plasmid.

How is the insulin beta chain produced?

The same process is repeated using the insulin beta gene, plasmid B, and Bacteria B.

How is functional insulin produced at the end of the process?

Bacteria multiply to produce insulin A and B chains. These are purified, beta-galactosidase is removed, and the chains are chemically joined to form functional insulin.

Outline the steps that are required for the human insulin gene to be cloned and expressed in bacteria.

1. isolate human insulin genes from human or artificially synthesise them from a known sequence through PCR

2. insulin alpha gene and plasmid are cut with EcoRI restriction enzyme placing the alpha gene next to beta galactosidase gene, repeat this for insulin beta gene in a second plasmid

3. DNA ligase is used to join the phosphodiester bonds in the plasmids and insulin genes to form two recombinant plasmid

4. the plasmids are placed into two separate plasmids via heat shock

5. the bacteria are blue/white screened to look for blue colonies, using beta galactosidase to indicate successful transformation

6. these successful bacteria are then grown in culture and replicate binary fission to produce large amounts of insulin fusion protein, which are isolated, joined together, and purified.

the three main bioethics approaches?

Consequence-based, duty/rule-based, and virtues-based (less commonly used).

key bioethical principles

Integrity, beneficence, non-maleficence, respect (less commonly used), and justice (less commonly used).

ethics

A system of moral principles concerned with what is beneficial to individuals and society, guiding how people live and make decisions.

consequence-based approach

Focuses on actions that lead to the best overall outcome for the most people, minimising negative effects, regardless of whether the action is 'right' or 'wrong'.

What action will lead to the best outcome overall?

duty/rule-based approach

Emphasises following rules, laws, or moral obligations, regardless of the consequences; often linked to professional or societal duties.

virtues-based approach

Focuses on the character and virtues of the person making the decision — what would a morally good person do?

integrity

Acting with honesty and trustworthiness; doing what is honest and ethically right.

justice

Fair distribution of the benefits and burdens of actions, ensuring no group is unfairly impacted or left out.

beneficence

Commitment to maximising benefits and minimising risks or harm when choosing a course of action.

What action is in the best interest of this patient or person?

non-maleficence

The principle of avoiding harm — actions should not cause harm, especially if the harm isn't outweighed by benefits.

respect

Recognising the intrinsic value and autonomy of living things; includes protecting those who can't make decisions for themselves.

What is CRISPR-Cas9?

"A defence mechanism found in bacteria to recognise and cut viral DNA (e.g. bacteriophage DNA). In biotechnology, it's used to target and edit specific DNA sequences using a guide RNA (gRNA) and the Cas9 nuclease."

What does CRISPR stand for?

Clustered Regularly Interspaced Short Palindromic Repeats.

What does Cas9 do in CRISPR-Cas9?

Cas9 binds to the PAM site and begins unzipping complementray DNA sequences and begins unzipping complementary DNA sequences. Cas9 will then cut the DNA several nucelotides away from the PAM site. also it can only bind to the pam site if the gRNA is complementary to the target DNA

What is a nuclease?

An enzyme that cuts DNA or RNA by breaking phosphodiester bonds between nucleotides.

What is a phosphodiester bond?

A bond that links the phosphate group of one nucleotide to the sugar of the next, forming the backbone of DNA or RNA.

What is gRNA?

Guide RNA that is complementary to a specific DNA sequence. It directs the Cas9 enzyme to the target DNA.

What are PAM sites?

Short DNA sequences (e.g. NGG) that must be adjacent to the target DNA for Cas9 to bind and cut. N = any nucleotide.

What are palindromic repeats in CRISPR?

"DNA sequences that read the same 5' to 3' on both strands; allows restriction enzymes to recognise and cut them."

What are spacers in CRISPR?

Stored viral DNA fragments from past infections, used by bacteria to recognise and defend against repeated attacks.

Steps of CRISPR-Cas9 (simple version)

1. gRNA binds Cas9 to form a complex 2. The complex binds to the PAM site 3. DNA is unzipped, and gRNA binds complementary DNA 4. Cas9 cuts the DNA several bases downstream of PAM

What can CRISPR do to DNA?

Delete: Remove DNA sections | Repair: Replace base pairs or insert genes | Mutate: Disrupt gene function

How is CRISPR used in biotechnology?

1. A synthetic gRNA is created to match the gene of interest 2. Cas9 enzyme is bound to this gRNA 3. The complex finds the PAM site and binds 4. Cas9 cuts the DNA 5. DNA may be repaired, deleted, or modified

Example: How CRISPR is used to improve rice (bacterial blight resistance)

1. SWEET gene exploited by Xanthomonas oryzae identified 2. gRNA and Cas9 designed to target SWEET gene 3. Cas9 cuts within the SWEET gene 4. CRISPR-edited rice becomes resistant to bacterial blight

How CRISPR can improve photosynthetic yield in plants

Modify Rubisco to prefer binding CO₂ over O₂; improve stress tolerance and grain quality, enhancing plant productivity

What are endonucleases and what do they do?

Restriction enzymes naturally found in bacteria that cut DNA at specific recognition sites to defend against viruses

What is DNA ligase and what does it do?

An enzyme that joins DNA fragments together by forming strong covalent bonds between them

What is DNA polymerase and what is its role?

An enzyme that adds free nucleotides to synthesize a complementary DNA strand during replication

What is RNA polymerase and what is its role?

An enzyme that adds RNA nucleotides to form a complementary mRNA strand during transcription

What is a palindromic restriction site?

A DNA sequence that reads the same 5' to 3' on both strands, allowing restriction enzymes to recognise and cut it

What are sticky ends in DNA fragments?

Overhangs of unpaired bases on one strand created by restriction enzymes, useful for combining DNA from different sources

What are blunt ends in DNA fragments?

DNA ends with no overhangs, produced by some restriction enzymes

Why do scientists prefer restriction enzymes that produce sticky ends?

Sticky ends make it easier to combine DNA from different sources due to complementary base pairing

Why do DNA fragments naturally anneal?

Because of hydrogen bonds forming between complementary base pairs, allowing fragments to stick together

Why do different restriction enzymes cut DNA at different places?

Each enzyme recognises and cuts at its own specific recognition sequence

How can restriction enzymes be used together?

They can be used alone or with others to cut DNA at multiple sites

What do restriction enzymes produce when cutting DNA?

Fragments of varying lengths depending on where they cut

What does DNA ligase do after restriction enzymes cut DNA?

It joins the fragments permanently using covalent bonds

What is the polymerase chain reaction (PCR)?

A method used to amplify small amounts of DNA rapidly in the lab

What is the purpose of PCR?

To create enough DNA for further analysis, such as gel electrophoresis

Why is Taq polymerase used in PCR?

Because it is heat-stable and doesn't denature at high temperatures required for PCR

What are the basic steps of PCR?

1. DNA, primers, free nucleotides, and Taq polymerase are mixed 2. Denaturation at ~95°C 3. Annealing at ~55°C 4. Extension at ~72°C, repeated to amplify DNA

What happens during denaturation in PCR?

DNA is heated to ~95°C to break hydrogen bonds and separate the strands

What happens during annealing in PCR?

DNA is cooled to ~55°C, and primers bind to the 3' ends of both DNA strands

What happens during extension in PCR?

Temperature is raised to ~72°C, Taq polymerase binds to primers and adds nucleotides to synthesize new DNA strands

What are primers and what do they do in PCR?

Short sequences that signal where Taq polymerase should begin replication

Why is PCR considered semi-conservative?

Each new DNA strand retains one original strand and synthesizes a new complementary one

What must be prevented during PCR and why?

Contamination, as it could lead to the amplification of unintended DNA and produce false results

What is reverse transcriptase and how is it used?

An enzyme from viruses (e.g. HIV) that converts RNA to DNA; in labs, it creates cDNA from RNA for PCR, e.g. in COVID-19 testing

What are the steps of gel electrophoresis?

1. Load DNA into gel wells 2. Apply electric current 3. DNA (negatively charged) moves to positive terminal 4. Shorter fragments move faster 5. Compare to DNA ladder

How does gel electrophoresis separate DNA?

By size and charge: smaller fragments move further through the porous gel; DNA moves to the positive terminal

What is DNA profiling?

Identifying individuals based on differences in their non-coding DNA regions

What are Short Tandem Repeats (STRs)?

areas of 2-6 repeating bases on our chromosomes that are used for profiling, we have 2 copies of each STR, one on each chromosome

How is DNA profiling done?

1. Collect DNA 2. Cut with restriction enzymes (if needed) 3. Amplify STRs with PCR 4. Use gel electrophoresis to compare samples

Why is DNA negatively charged?

Because of the phosphate groups in its backbone

What can CRISPR do?

create a synthetic guide RNA (sgRNA) to target the area of the genome you want to edit, delete, repair, or mutate DNA.

How does CRISPR delete DNA?

by removing a section of DNA.

How does CRISPR repair DNA?

by replacing base pairs or inserting a new gene.

How does CRISPR mutate DNA?

by disrupting a gene to prevent it from functioning.

What is CRISPR-Cas9?

a defense mechanism found in bacteria that remembers bacteriophages that are trying to infect the bacteria, helping to protect them.

Where is CRISPR found?

in bacteria, and it contains DNA from previous bacteriophage infections that have been stored.

What is a bacteriophage?

A a virus that infects and replicates itself within bacteria.

What is Cas9?

a nuclease enzyme used in CRISPR technology to cut DNA at a specific site, such as viral or target gene sequences.

What is a nuclease?

an enzyme that cuts DNA or RNA by breaking the bonds between nucleotides.

What is a phosphodiester bond?

a bond that links the sugar of one nucleotide to the phosphate of the next, forming the backbone of DNA or RNA.

What does g-RNA do?

it allows the CRISPR-Cas9 complex to identify a specific DNA sequence in the genome, where Cas9 can then cut the DNA.

What does CRISPR stand for?

Clustered Regularly Interspaced Palindromic Repeats.

What is a spacer in CRISPR?

a stored bacteriophage DNA sequence from previous encounters