Chapter 2
Host–Vector System
EXERCISES
1. Describe the importance of host vector system in rDNA technology.
Answer: In recombinant DNA (rDNA) technology, the host vector system plays a critical role by bridging the gap between isolated genes and their functional expression. It’s like a tiny delivery system that carries the gene of interest into a living cell and provides the machinery necessary for its replication and expression. Here’s why the host vector system is so important:
1. Gene Delivery:
Isolated genes, even if identified and purified, cannot simply enter a cell and function on their own. The host vector, typically a plasmid or a virus, serves as a vehicle, carrying the gene of interest into the host cell.
The vector is engineered to have specific features that facilitate entry into the host cell, either by natural mechanisms or through engineered processes.
2. Gene Replication:
*Once inside the host cell, the vector replicates alongside the host’s own DNA, ensuring the gene of interest is also copied and maintained. This prevents the loss of the introduced gene over time.
*Some vectors integrate into the host chromosome for stable inheritance, while others remain as separate entities replicating autonomously.
3. Gene Expression:
*Besides carrying the gene, the vector also possesses regulatory elements like promoters and enhancers that control the expression of the gene of interest.
*These elements signal to the host cell’s machinery to transcribe the gene into mRNA and translate it into the desired protein product.
4. Host Compatibility:
*The choice of host and vector is crucial for successful rDNA technology. The host cell should be compatible with the vector, able to efficiently take it up, replicate it, and express the encoded gene.
*Common host organisms include bacteria like E. coli and yeast, while the specific vector type can be chosen based on desired features like copy number, expression levels, and ease of manipulation.
5. Scalability and Control:
*Host vector systems allow for the production of large quantities of the desired protein product, making them invaluable for research and therapeutic applications.
*Additionally, elements within the vector can be modified to fine-tune the expression levels or control the timing of protein production.
*In summary, the host vector system acts as the backbone of rDNA technology, playing a vital role in every step from gene delivery to protein expression. Its versatility, scalability, and controllability make it an essential tool for various fields, including:
*Biomedical research: Understanding gene function, developing new diagnostic tools, and testing potential drug targets.
*Production of therapeutic proteins: Insulin, hormones, and enzymes for treating various diseases.
*Biotechnology applications: Production of industrial enzymes, biofuels, and other valuable products.
The constant development and improvement of host vector systems continue to expand the possibilities of rDNA technology, driving innovation in both basic science and applied research.
2. What are the major characteristics of a vector?
Answer: Vectors have 3 key characteristics:
*Magnitude and direction: Like an arrow, they have both size and pointing direction.
*Addition and subtraction: Follow specific rules to combine or remove their effects.
*Scaling: Can be stretched or shrunk by multiplying by a number.
These traits make vectors essential for representing and manipulating various concepts in math, physics, and biology.
3. What is plasmid and what are its different types?
Answer: A plasmid is a tiny, circular DNA molecule that lives inside some cells (mostly bacteria) but isn’t part of their main chromosomes. They come in different types like antibiotic resistance plasmids, virulence plasmids, and more. These types help bacteria survive in harsh environments, become harmful, or just hang around without doing much. Plasmids are super useful in biotechnology too, helping scientists move genes around like tiny DNA taxis!
4. Discuss the strategy applied for the development of (pBR322) plasmid cloning vectors.
Ans: The pBR322 plasmid was a landmark development in the field of plasmid cloning vectors. Its design incorporated several key strategies to achieve its functionality and versatility:
1. Replicon:
*pBR322 contains a high-copy replicon derived from the ColE1 plasmid. This allows for the plasmid to replicate autonomously within the host cell at a high copy number, ensuring ample availability of the cloned gene for subsequent analysis or expression.
2. Selectable Markers:
*The plasmid features two antibiotic resistance markers, one for ampicillin and the other for tetracycline. This allows for selection of transformed cells harboring the plasmid by plating them on media containing the corresponding antibiotic. Cells without the plasmid will be eliminated due to antibiotic sensitivity.
3. Multiple Cloning Site (MCS):
pBR322 contains a strategically placed MCS with unique restriction enzyme recognition sites. These sites allow for the insertion of foreign DNA fragments at a defined location within the plasmid without disrupting its essential functions.
4. Plasmid Size and Stability:
*pBR322 is relatively small in size (approximately 4361 base pairs) compared to the host bacterial genome. This minimizes the burden on the host cell and reduces potential instability issues associated with large plasmids.
5. Flexibility and Compatibility:
*The features of pBR322 are designed for broad compatibility with various bacterial hosts commonly used in cloning experiments. This flexibility allows researchers to choose the ideal host-vector combination for their specific needs.
6. Additional Features:
*pBR322 also includes the origin of replication and RNA polymerase promoter for the lacZ gene, enabling convenient blue/white color screening for the presence of inserts within the MCS.
Impact and Legacy:
*pBR322, despite its limitations, served as a foundational technology for decades, influencing the design of countless future plasmid vectors. Its simple, effective design principles and modular architecture paved the way for a diverse range of specialized vectors with advanced features to cater to specific cloning, expression, and manipulation needs.
In summary, the development of pBR322 relied on a synergistic combination of strategies, including high-copy replication, selectable markers, a dedicated cloning site, appropriate size and stability, and versatile design, making it a cornerstone in the history of plasmid cloning vectors.
5. Briefly describe the structure of lambda bacteriophage and also discuss the role of lambda phage based vectors.
Ans: Lambda Bacteriophage Structure and Lambda Phage Vectors
Structure:
*Head: Icosahedral head containing the 48,502 bp double-stranded DNA genome.
*Tail: Long, flexible tail used for attaching to and injecting DNA into the host cell.
*Fibers: Six tail fibers that recognize specific receptors on the host cell surface.
Functional Domains:
*Left and Right Arms: Flanking regions of the DNA with homologous sequences used for integration into the host chromosome during the lysogenic cycle.
*Central Core: Contains genes for replication, transcription, and assembly of phage particles.
*Cos Sites: Cohesive ends of the DNA molecule necessary for circularization in the host cell.
*Regulatory Elements: Promoters and operators controlling gene expression and switching between the lytic and lysogenic cycles. Lambda Phage Vectors: *Derived from the lambda phage genome: Engineered vectors retaining essential elements for packaging and delivery of foreign DNA. Features:
*Cos sites: Allow for efficient packaging of foreign DNA into lambda phage particles.
*Selectable markers: Antibiotic resistance genes for identification of transformed cells.
*Cloning sites: Specific restriction enzyme recognition sites for insertion of foreign DNA.
*Promoters and regulatory elements: Facilitate expression of the cloned gene.
Uses:
*Gene cloning: Efficiently amplify and propagate large DNA fragments.
*Genome mapping: Create physical maps of large genomes. *Mutagenesis: Introduce mutations into genes for functional studies.
*Gene therapy: Delivering therapeutic genes to specific cells. Advantages:
*High packaging capacity: Can package up to 23 kb of foreign DNA.
*Efficient delivery: High infectivity for bacterial cells.
*Versatile features: Adaptable for various applications.
Limitations:
*Size restriction: Large inserts can reduce packaging efficiency.
*Specificity: Requires compatible host cells for efficient infection.
Overall, lambda phage vectors are powerful tools in recombinant DNA technology and genomics, offering efficient DNA delivery and flexibility for diverse research applications.
6. Discuss the M13 based vectors and its application.
Answer: Here’s a short answer on M13-based vectors and their applications:
M13 vectors are derived from a filamentous bacteriophage (virus that infects bacteria) called M13. They offer unique advantages for certain molecular biology applications:
Key features:
*Single-stranded DNA: M13 replicates as single-stranded DNA, making it ideal for sequencing and mutagenesis studies.
*High yields: M13 vectors produce large amounts of DNA, useful for protein expression and structural studies.
*Phage display: Allows fusion of proteins or peptides to the phage coat, enabling selection and screening of specific molecules.
Applications:
*Sequencing: Facilitates direct sequencing of DNA inserts without cloning steps. *Site-directed mutagenesis: Convenient introduction of specific mutations into DNA sequences.
*Protein expression: Production of proteins in their native, soluble form for structural and functional studies. *Phage display: Screening for high-affinity antibodies, drug targets, and protein-protein interactions.
*Nanotechnology: Production of nanowires for use in electronics and materials science.
In summary, M13 vectors provide valuable tools for sequencing, mutagenesis, protein production, screening, and nanotechnology applications, making them versatile assets in molecular biology research.
7. Differentiate between cosmids and phagemids.
Answer: Both cosmids and phagemids are valuable tools in molecular biology, but they have distinct characteristics and applications. Here’s a breakdown of their key differences:
Detailed explanation:
*DNA Source: Cosmids are hybrid vectors, combining features of plasmids and lambda phage. They contain a cos site from lambda phage, allowing them to integrate into the host chromosome during the lysogenic cycle. Phagemids, on the other hand, are modified plasmids containing an F1 phage origin of replication. This enables them to be packaged into phage particles under specific conditions.
*Replication: Both cosmids and phagemids can replicate autonomously within the host cell like plasmids. However, phagemids have the additional ability to be packaged into infectious phage particles, facilitating efficient delivery into new host cells.
*DNA Size Limit: Cosmids, due to their hybrid nature, can accommodate larger DNA inserts (35-52 kb) compared to phagemids (around 20 kb). This makes them ideal for cloning bigger genomic fragments.
*Delivery Method: Cosmids exclusively rely on transformation for introducing them into host cells. Phagemids, on the other hand, can be introduced through both transformation and phagemid particles. The latter mode allows for rapid infection and propagation in bacterial cultures.
*Applications: Cosmids are primarily used for cloning larger DNA fragments, essential for creating genomic libraries or generating large plasmids. Phagemids find application in gene expression libraries and phage display. Their ability to form phage particles helps create libraries of expressed proteins for functional studies, while phage display utilizes the phage coat for displaying and selecting specific sequences or binding partners.
*Advantages and Disadvantages: Cosmids offer the advantage of high capacity and stability, but their large size can limit delivery efficiency. Phagemids provide efficient delivery through phage particles and are suitable for expression libraries, yet their smaller capacity and potential instability are drawbacks. Choosing between cosmids and phagemids depends on the specific research needs. For cloning large fragments, cosmids are preferred. For gene expression libraries and phage display, phagemids offer distinct advantages.
8. Why is a vector required for cloning of a gene?
Answer: Vectors are vital for gene cloning because they provide a carrier molecule for the foreign gene to be introduced and propagated in a host cell. They offer several crucial functions:
1. Delivery: Vectors have mechanisms to enter the host cell efficiently, overcoming natural barriers that would prevent the naked gene from entering and establishing itself.
2. Replication: Vectors possess their own replication machinery or utilize the host’s machinery to amplify the cloned gene along with their own DNA, ensuring multiple copies are produced for further analysis or applications.
3. Selection: Most vectors contain markers like antibiotic resistance genes, allowing for identification and selection of cells that have successfully been transformed with the vector and the gene of interest.
4. Expression: Certain vectors include control elements like promoters and enhancers to drive the expression of the cloned gene, turning its DNA sequence into functional protein products.
5. Stability: Vectors ensure the stable maintenance of the cloned gene within the host cell, preventing its loss or degradation over time.
In essence, vectors act like tiny vehicles, packaging the gene of interest, delivering it into the host cell, providing the necessary machinery for its replication and expression, and offering tools for selection and maintenance. Without these capabilities, a naked gene wouldn’t be able to effectively enter, replicate, express, or persist within a host cell, making gene cloning impossible.
9. A plasmid capable of getting integrated into host chromosome is called:
(a) Col plasmid
(b) Episome
(c) Ti plasmid
(d) R plasmid
Ans: (b) Episome.
10. Why the replication of single copy plasmid called stringent replication?
Ans: You’re right! Single-copy plasmids, where only one or a few copies exist per host cell, are often referred to as stringent plasmids due to the tight control mechanisms governing their replication. This contrasts with relaxed plasmids, which replicate at a higher rate, resulting in many copies per cell.
Here’s why stringent replication is essential for single-copy plasmids:
1. Maintaining Copy Number: Stringent control ensures the plasmid copy number remains within a narrow range, preventing over-replication. Too many plasmid copies can be detrimental to the host cell, burdening its resources and potentially altering its metabolic processes.
2. Stability and Inheritance: By avoiding over-replication, stringent control minimizes plasmid loss during cell division. This ensures each daughter cell receives a copy of the plasmid, promoting stable inheritance.
3. Regulated Expression: In some cases, the gene(s) encoded by the single-copy plasmid may have a strong impact on host cell function. Stringent control ensures the expression level of these genes remains manageable, preventing disruption of vital cellular processes.
Mechanisms of Stringent Control:
The specific mechanisms underlying stringent control vary between plasmids, but some common examples include:
*Positive control: Activator proteins bind to specific sites on the plasmid DNA, initiating replication at controlled intervals.
*Negative control: Repressor proteins bind to the origin of replication, blocking initiation until certain conditions are met (e.g., availability of specific nutrients).
*Specific incompatibility systems: Plasmids incompatible with each other trigger degradation of one or both plasmids, preventing over-accumulation.
Importance of Stringent Replication:
Stringent control of single-copy plasmid replication is crucial for maintaining a healthy balance between the plasmid and the host cell. It ensures stable inheritance, allows for regulated gene expression, and prevents detrimental effects on the host’s physiology.
11. Identify the incorrect match pair from the following:
(i) Multi copy plasmid (a) Stringent replication
(ii) Col plasmid (b) Kills bacteria
(iii) pBR322 (c) Plasmid
(iv) Prophage (d) Phage genome inserted into a host genome
Ans: (i) Multi copy plasmid (a) Stringent replication.
12. How can a large size eukaryotic gene insert be cloned?
Ans: Cloning large eukaryotic gene inserts (greater than 20 kb) presents challenges due to size limitations of commonly used vectors like plasmids. However, several methods can overcome these limitations:
1. BAC (Bacterial Artificial Chromosome) vectors:
*Large (up to 300 kb) vectors derived from bacterial chromosomes, capable of accommodating large eukaryotic inserts.
*Stable and easy to manipulate in bacteria.
*Useful for creating genomic libraries and large-scale sequencing projects.
2. YAC (Yeast Artificial Chromosome) vectors:
Even larger (up to 2 Mb) vectors derived from yeast chromosomes.
*Can accommodate even larger inserts than BACs.
*Less stable and more complex to manipulate than BACs.
(Suitable for cloning very large genomic regions or complex gene clusters.
3. Cosmid vectors:
*Smaller than BACs and YACs (up to 50 kb) but larger than traditional plasmids.
*Can accommodate moderately large inserts.
*Easier to manipulate than BACs and YACs.
*Useful for cloning genes or genomic regions too large for plasmids but too small for BACs.
4. In vitro cloning methods:
*Techniques like homologous recombination in cell-free systems allow direct assembly of large DNA fragments without using vectors.
*Offer greater flexibility and potentially overcome size limitations.
*Still under development and may require specialized expertise.
Choosing the best method depends on the specific size of the gene insert, desired level of stability and ease of manipulation, and available resources.
Here are some additional factors to consider:
*Complexity of the insert: Repetitive sequences within the insert can pose challenges for certain cloning methods. Availability of suitable restriction sites: Proper restriction sites are needed for efficient insertion into the vector. .
* downstream applications:** Certain methods might be better suited for specific applications like functional studies or protein expression.
13. Assertion: An ideal vector should have selectable marker.
Reason: Selectable markers are required to screen out transformation.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false.
Ans: (b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
14. Assertion: Cosmid is a hybrid vector.
Reason: Cosmid has properties of both plasmids and lambda phage vector.
(a) Both assertion and reason are true and the reason is the correct explanation of the assertion.
(b) Both assertion and reason are true but the reason is not the correct explanation of the assertion.
(c) Assertion is true but reason is false.
(d) Both assertion and reason are false
Ans: (a) Both assertion and reason are true and the reason is the correct explanation of the assertion.