explain that genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism and that this may involve transferring a gene into an organism so that the gene is expressed

Principles of Genetic Technology (Cambridge 9700 – Topic 19)

Learning Objective (AO1)

Genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism. This usually involves transferring a gene into a host cell so that the gene is expressed, giving the host a new trait.

Key Definitions

• Genetic engineering (genetic modification): Intentional alteration of an organism’s DNA to obtain a desired characteristic.
• Gene: A DNA segment that encodes a functional product (protein or functional RNA).
• Vector: A DNA molecule (plasmid, virus, or artificial construct) that carries a foreign gene into a host cell.
• Host organism (host cell): The cell or organism that receives the recombinant DNA and expresses the introduced gene.
• Promoter: DNA sequence upstream of a gene that recruits RNA polymerase and initiates transcription; must be recognised by the host’s transcription machinery.
• Terminator: Sequence downstream of a gene that signals transcription termination and, in eukaryotes, provides a poly‑A signal.
• Selectable marker: Gene conferring a detectable trait (e.g., antibiotic resistance, fluorescent protein) that allows identification of cells that have taken up the vector.
• Expression: The process by which a gene’s information is transcribed into RNA and translated into a functional product.
• Origin of replication (ori): DNA region that allows the vector to replicate independently in the host.
• Ribosome‑binding site (RBS) / Kozak sequence: Sequence that ensures efficient translation initiation in prokaryotes (RBS) or eukaryotes (Kozak).

Regulatory & Safety Frameworks (AO1)

Typical Recombinant‑DNA Workflow (AO1 & AO2)

1. Design of the construct

   • Choose a promoter appropriate for the host (e.g., T7 for E. coli, CaMV 35S for plants).
   • Select a terminator (e.g., T7 terminator, NOS terminator).
   • Decide on a selectable marker (amp^R, hygromycin‑B phosphotransferase, GFP).
   • Include an origin of replication that works in the host (pMB1 ori for bacteria, 2 µ plasmid ori for yeast).
   • Add a RBS (prokaryotes) or Kozak sequence (eukaryotes) to ensure efficient translation.

2. Isolation of the gene of interest

   • Extract genomic DNA from the donor organism.
   • Cut the DNA with specific restriction enzymes (e.g., EcoRI, HindIII) that create compatible sticky or blunt ends.

3. Construction of the recombinant vector

   • Linearise the chosen vector with the same restriction enzymes used for the gene.
   • Ligate the gene fragment into the vector using T4 DNA ligase (16 °C overnight or 20 °C for 1 h, with ATP and Mg²⁺).
   • Verify that the promoter, terminator, selectable marker and ori are present in the final construct.

4. Introduction of the recombinant vector into the host

   • Transformation* (chemical CaCl₂ or electroporation) – bacteria.
   • *Transfection* (liposome, calcium phosphate) – eukaryotic cell lines.
   • *Microinjection* – animal embryos.
   • *Agrobacterium‑mediated transfer* – dicot plants (engineered strains also work in monocots).
   • *Viral vectors* – adenovirus, lentivirus for animal cells.
   • *CRISPR‑Cas9* – direct genome editing; plasmid‑delivered Cas9/gRNA follows the same steps.

5. Selection of successfully modified cells

   • Plate on medium containing the appropriate antibiotic or apply the relevant selective pressure.
   • Optional: use a reporter gene (e.g., GFP) to visualise transformed colonies under UV light.

6. Verification of gene insertion and expression (AO2)

   • Restriction‑digest analysis: isolate plasmid DNA, cut with diagnostic enzymes, run on agarose gel, compare band pattern with expected sizes.
   • Polymerase‑chain reaction (PCR): amplify the inserted gene; confirm size by gel electrophoresis.
   • Sequencing (optional) to verify orientation and absence of mutations.
   • Expression assays:

     • RT‑PCR or Northern blot for mRNA.
     • Western blot, ELISA, or activity assay for protein (e.g., insulin bioassay).

Common Sources of Error (AO2 – Evaluation)

• Incomplete restriction‑enzyme digestion → mixed vector population.
• Low ligation efficiency caused by sub‑optimal ATP/Mg²⁺ concentration or inappropriate temperature.
• Pipetting inaccuracies when adding small volumes of DNA or enzymes.
• Contamination of competent cells with non‑competent cells, reducing transformation efficiency.
• Gel electrophoresis artefacts (smiling bands, diffusion) that hinder accurate size estimation.
• Incorrect colony counting (over‑crowding, missed small colonies) affecting transformation‑efficiency calculations.

Components Required for Gene Expression (AO1)

• Promoter – recognised by host RNA polymerase (e.g., T7 for *E. coli*, CaMV 35S for plants).
• Ribosome‑binding site (RBS) / Kozak sequence – ensures efficient translation initiation.
• Terminator – stops transcription and, in eukaryotes, provides a poly‑A signal.
• Selectable marker – enables identification of transformed cells (antibiotic resistance, fluorescent protein).
• Origin of replication (ori) – allows independent replication of the vector in the host.

Practical Data‑Analysis Skills (AO2)

1. Interpreting a Restriction‑Digest Gel

Example: A 5 kb plasmid cut with EcoRI (single site) and a 1.2 kb insert also cut with EcoRI.

2. Calculating Transformation Efficiency

Formula: Efficiency (cfu µg⁻¹ DNA) = (Number of colonies × Dilution factor) ÷ (µg of DNA used)

Example calculation:

• Plated 100 µL of a 1 : 1000 dilution.
• Counted 45 colonies.
• DNA used = 0.05 µg.

Efficiency = (45 × 1000) ÷ 0.05 = 9 × 10⁵ cfu µg⁻¹.

3. Critical Evaluation of Techniques (AO2)

Applications of Genetic Engineering (AO1)

• Therapeutic proteins: Human insulin produced in E. coli (commercial product Humulin).
• GM crops: Bt corn (expresses *Bacillus thuringiensis* toxin for insect resistance); Golden Rice (engineered to produce β‑carotene).
• Gene therapy: Adeno‑associated virus (AAV) vector delivering a functional RPE65 gene for Leber congenital amaurosis.
• Model organisms: Knock‑out mouse lacking the p53 tumour‑suppressor gene to study cancer pathways.
• Industrial enzymes: Recombinant cellulase expressed in *Trichoderma reesei* for bio‑fuel production.

Ethical, Social & Environmental Issues (AO1)

• Gene flow from GM crops to wild relatives → possible loss of biodiversity.
• Unintended health effects (allergenicity, unknown toxins).
• Socio‑economic concerns: patents on life forms, farmer dependence on proprietary seed companies.
• Dual‑use concerns: Gene‑drive technology could be used to suppress disease‑vector mosquitoes, but the same approach could be mis‑applied to spread harmful traits in wild populations.
• Need for robust regulatory oversight to balance innovation with safety.

Mini‑Case Study (AO3 – Planning, Execution, Evaluation)

Task: Design a CRISPR‑Cas9 experiment to knock‑out the *phytoene desaturase* (PDS) gene in *Arabidopsis thaliana* to obtain albino seedlings.

1. Objective – Disrupt PDS, a key enzyme in carotenoid biosynthesis, producing a visible phenotype that confirms successful editing.
2. Materials & Reagents

   • pHEE401E vector (Cas9 under 35S promoter, sgRNA scaffold).
   • sgRNA oligonucleotides targeting exon 1 of PDS.
   • Agrobacterium tumefaciens strain GV3101.
   • Selective medium containing hygromycin.

3. Experimental Steps

   1. Design two 20‑nt sgRNA sequences flanking a 20‑bp region of exon 1; check off‑target sites using a web‑based tool.
   2. Clone sgRNA duplexes into pHEE401E using BbsI sites.
   3. Transform the construct into *A. tumefaciens* (electroporation).
   4. Perform floral‑dip transformation of *A. thaliana* (Col‑0) plants.
   5. Harvest T₁ seeds, surface‑sterilise, and sow on hygromycin‑containing plates.
   6. Screen hygromycin‑resistant seedlings for albino phenotype.
   7. Confirm mutation by PCR amplification of the target region followed by Sanger sequencing.

4. Controls

   • Negative control: plants dipped in *A. tumefaciens* carrying an empty vector.
   • Positive control: use a previously validated sgRNA that produces a known phenotype.

5. Evaluation Criteria (AO2)

   • Transformation efficiency: number of hygromycin‑resistant seedlings per 100 seeds.
   • Editing efficiency: proportion of albino seedlings among resistant plants.
   • Specificity: sequencing data showing only the intended indel and no off‑target mutations.

6. Potential Sources of Error (AO2)

   • Poor sgRNA design → low cutting efficiency.
   • Incomplete Agrobacterium infection during floral dip.
   • Chimeric T₁ plants leading to mosaic phenotypes.

Suggested Diagram

Flowchart of the genetic‑engineering process:

1. Identify & design construct (promoter, terminator, marker, ori, RBS/Kozak)
2. Isolate target gene
3. Cut gene & vector with compatible restriction enzymes
4. Ligate gene into vector
5. Introduce recombinant vector into host (transformation, transfection, etc.)
6. Select transformed cells
7. Verify insertion (gel, PCR, sequencing)
8. Confirm expression (RNA/protein assay)
9. Scale‑up / application

Quick‑Check Questions (Revision)

1. Define “promoter” and give an example of a promoter used in plant genetic engineering.
2. Why must restriction enzymes that generate compatible ends be used when ligating a gene into a plasmid?
3. Calculate the transformation efficiency if 120 colonies are obtained from 0.2 µg of plasmid DNA plated at a 1 : 500 dilution.
4. List two advantages and two limitations of CRISPR‑Cas9 compared with traditional recombinant‑DNA methods.
5. Explain one ethical concern associated with the release of genetically modified crops.

Structured‑question Style Item (AO2)

Question: Explain how you would confirm that a transgenic *Arabidopsis* plant expresses the introduced *gusA* (β‑glucuronidase) gene. In your answer, describe at least two different experimental approaches and the type of result that would indicate successful expression.