explain why a promoter may have to be transferred into an organism as well as the desired gene

Principles of Genetic Technology – Cambridge AS & A Level Biology (9700)

Objective

Explain why a promoter may have to be transferred into an organism together with the desired gene, and place this requirement within the full context of recombinant‑DNA technology.

Syllabus Scope (Topic 19 – Genetic Technology)

• Restriction enzymes, ligation and the creation of recombinant DNA.
• Vectors (plasmids, viral vectors, plant‑transformation vectors, BAC/YAC).
• Transformation / transfection techniques.
• Selection, screening **and** verification of recombinant clones.
• Gene‑expression control – promoters, terminators, enhancers.
• Applications (insulin, Bt toxin, herbicide‑resistant crops, gene therapy, CRISPR‑Cas9, etc.).
• Ethical, social and environmental considerations.

Key Definitions (AO 1)

Gene

A DNA segment that encodes a functional product, usually a protein.

Promoter

A DNA sequence upstream of a coding region that binds RNA polymerase and transcription factors to start transcription.

Terminator

A DNA sequence downstream of the coding region that signals the end of transcription and promotes release of the nascent RNA.

Enhancer

A DNA element that can be located upstream, downstream or within an intron; it increases transcription rates by binding activator proteins and looping to the promoter.

Vector

A DNA molecule (plasmid, virus, artificial chromosome) used to deliver foreign genetic material into a host cell.

Multiple Cloning Site (MCS)

A short stretch of DNA containing several unique restriction sites, usually placed downstream of a promoter.

Selectable marker

A gene (e.g., antibiotic‑resistance, herbicide‑resistance) that allows only cells which have taken up the vector to survive under selective conditions.

Reporter gene

A gene whose product is easy to detect (e.g., lacZ, GFP) and is used to monitor successful cloning or expression.

Host range

The spectrum of organisms that a particular vector can infect or be maintained in.

Transformation / Transfection

Introduction of recombinant DNA into a bacterial cell (transformation) or a eukaryotic cell (transfection).

Recombinant DNA Technology – The Complete Pipeline

1. Gene Isolation & Preparation

• Extract genomic DNA or mRNA from the donor organism.
• If starting from mRNA, reverse‑transcribe to obtain cDNA.
• Amplify the target gene by PCR, adding restriction sites to the ends if required.

2. Restriction‑Enzyme Digestion & Ligation

• Select restriction enzymes that cut both vector and insert to give compatible sticky or blunt ends.
• Ligate the insert into the vector’s MCS using DNA ligase, creating a recombinant DNA molecule.

3. Vector Types & Their Key Features

4. Gene‑Expression Control Elements

• Promoters – determine whether transcription occurs and how strongly.
• Terminators – ensure proper termination and polyadenylation (eukaryotes) or rho‑independent/dependent termination (prokaryotes).
• Enhancers – increase transcription rates; may act at a distance and are often tissue‑ or development‑specific.

5. Why Transfer a Promoter with the Gene?

• Species‑specific transcription factors: The donor gene’s native promoter may require factors absent in the host, leading to no transcription.
• Chromatin context (eukaryotes): Histone modifications and DNA methylation differ between species, affecting promoter accessibility.
• Regulatory signals: A promoter that responds to environmental cues not present in the host will remain silent.
• Desired expression level: Commercial or experimental aims often need high, constitutive, or tightly inducible expression that the native promoter cannot provide.
• Safety and control: An inducible promoter allows expression to be turned on only after the host cells have reached sufficient density, preventing toxicity.

Consequently, the gene is usually cloned downstream of a promoter that is known to function efficiently in the chosen host.

6. Common Promoter Choices

7. Transformation / Transfection Methods

8. Selection, Screening & Verification

• Antibiotic resistance – e.g., ampicillin, kanamycin; only cells that have taken up the vector survive.
• Reporter genes – lacZ (blue/white screening), GFP (fluorescence), luciferase (luminescence).
• Colony PCR / Restriction analysis – rapid confirmation of insert presence and orientation.
• Sequencing – final verification of the exact nucleotide sequence.

9. Applications (Illustrative Examples)

Ethical, Social & Environmental Issues (AO 1)

• Safety of GM organisms – gene flow to wild relatives, impact on biodiversity.
• Human health concerns – allergenicity, horizontal gene transfer.
• Intellectual property – patents on genes, seeds, and technologies.
• Equity of access – availability of life‑saving therapies in low‑income countries.
• Regulatory frameworks – EU GMO Directive, US FDA/USDA oversight, WHO guidelines.

AO 2 – Applying & Evaluating Information (Practice Task)

Task: The data below were obtained after transforming *E. coli* with a recombinant plasmid that contains the human growth‑hormone (hGH) gene downstream of either the native human promoter or the bacterial lac promoter. Cultures were grown with and without IPTG induction, and hGH concentration in the culture medium was measured by ELISA.

1. Interpret the results. Which construct gives the highest level of hGH and why?
2. Explain why the promoter choice is critical for commercial protein production.
3. Suggest two modifications that could further increase hGH yield in this system.

Marking guide (summary)

• Recognition that the lac‑promoter construct with IPTG gives ~64‑fold higher expression than the native promoter (≈13 µg mL⁻¹ vs ≤0.3 µg mL⁻¹). Reason: bacterial transcription machinery recognises the lac promoter; IPTG removes LacI repression, allowing strong transcription.
• Link to commercial needs – high yield reduces cost; inducible systems prevent toxicity during cell growth.
• Possible improvements: (i) use a high‑copy‑number plasmid; (ii) optimise codon usage for *E. coli*; (iii) co‑express a molecular chaperone or foldase; (iv) switch to a T7‑based system for even higher expression.

Key Points to Remember (AO 1 Summary)

• Promoters are essential for transcription; without a functional promoter the transferred gene remains silent.
• When moving a gene between species, the donor promoter often fails because transcription factors, chromatin context, and regulatory signals differ.
• Terminators ensure proper transcription termination; enhancers can dramatically boost expression and may confer tissue‑specificity.
• Choosing a promoter (and associated regulatory elements) compatible with the host guarantees efficient gene expression.
• The full recombinant‑DNA workflow includes: restriction‑enzyme digestion → ligation → vector selection → insertion of appropriate promoter/terminator/enhancer → transformation → selection/screening → verification → application.
• Ethical and safety considerations must be evaluated for every genetic‑technology project.

Summary

Transferring a promoter together with the desired gene guarantees that the host’s transcriptional machinery can recognise and efficiently transcribe the gene. The promoter (and, where relevant, terminator and enhancer) must be selected on the basis of host compatibility, required expression level, and whether expression should be constitutive, inducible, or tissue‑specific. This step is integral to the broader recombinant‑DNA pipeline prescribed by the Cambridge AS & A Level Biology syllabus, underpinning successful applications from insulin production to CRISPR‑mediated genome editing while also raising important ethical and societal questions.