Table of Contents
Bacteriophages – viruses that infect bacteria – are central tools in genetic engineering. In this chapter, the focus is on what is specific to phages as genetic engineering tools, not on viruses in general or on basic DNA structure (covered elsewhere).
What Are Bacteriophages in the Context of Genetic Engineering?
Bacteriophages (often shortened to “phages”) are:
- Obligate parasites of bacteria: They cannot reproduce by themselves; they must infect a bacterial cell.
- Natural DNA (or RNA) injectors: They have evolved efficient mechanisms to introduce their genetic material into bacteria.
- Models and tools: Much of molecular genetics and many genetic engineering methods originated from studying phages.
In genetic engineering, phages are used or studied mainly because:
- They transfer DNA between bacteria (important for understanding horizontal gene transfer).
- Their genomes are relatively small and well-characterized.
- Their life cycles can be controlled and exploited to introduce or amplify foreign DNA.
General Structure of Bacteriophages (Relevant Features)
While phage shapes are diverse, many well-studied phages (like bacteriophage $\lambda$ and T4) share some typical structural features that are important for genetic engineering:
- Head (capsid): Protein shell containing the nucleic acid (often double-stranded DNA).
- Tail (in many phages): A hollow structure that binds to the bacterial surface and injects the DNA.
- Tail fibers: Recognize and bind specific receptors on the bacterial cell wall or membrane, determining host specificity.
For genetic engineering, two structural features are crucial:
- Genome packaging capacity: How much DNA can fit into the head. This sets an upper and lower size limit for any inserted foreign DNA.
- Attachment and injection apparatus: A natural “syringe” that can deliver DNA into the bacterial cytoplasm with high efficiency.
Two Main Types of Phage Life Cycles: Lytic vs. Lysogenic
The life cycle type is decisive for how a phage can be used in genetic engineering.
Lytic Phages
- After infection, the phage takes over the bacterial metabolism.
- It replicates its DNA, produces phage proteins, assembles new phage particles.
- Eventually, the bacterium is lysed (broken open), releasing many new phages.
Key points for genetic engineering:
- Rapid amplification: Lytic phages can produce large numbers of copies of a DNA molecule in a short time.
- Plaque formation: Lysis causes clear zones (“plaques”) in a bacterial lawn, which can be used to isolate and count phage particles.
- Lytic phages can be used as cloning and screening tools, because each plaque originates from a single phage and thus from a single DNA molecule.
Temperate Phages and Lysogeny
Temperate phages (such as bacteriophage $\lambda$) can follow two alternative pathways after infection:
- Lytic cycle (as above), or
- Lysogenic cycle:
- Phage DNA integrates into the bacterial chromosome or persists as a stable plasmid-like element.
- The integrated phage DNA, now called a prophage, is replicated passively with the bacterial chromosome.
- The host cell is “lysogenized” and usually not lysed as long as the prophage remains dormant.
Switching from lysogeny to lysis can be triggered by certain conditions (e.g., DNA damage).
For genetic engineering, lysogenic phages are valuable because:
- They can act as vectors that integrate foreign DNA into the bacterial genome.
- They allow stable maintenance of inserted genes over many bacterial generations.
- Their life cycle is highly regulated, providing control over when inserted genes are expressed or excised.
Bacteriophage $\lambda$ as a Model and Tool
Among the many known phages, bacteriophage $\lambda$ (lambda) is the best-known genetic engineering tool in bacteria.
Basic Properties (Relevant for Genetic Engineering)
- Infects Escherichia coli (E. coli).
- Double-stranded linear DNA genome of about 48.5 kilobase pairs (kb).
- Can be lytic or lysogenic.
- Has defined DNA packaging signals and non-essential regions, which make it modifiable.
Important features used in genetic engineering:
- Cos sites:
- Short DNA sequences at the ends of the $\lambda$ genome.
- Allow the phage DNA to be packaged into capsids as a linear molecule.
- When inside the bacterium, the complementary cos ends can anneal and be ligated to form a circular DNA.
- These cos sequences are exploited in “cosmids” (hybrid plasmid–phage vectors) to package large DNA fragments.
- Non-essential genes:
- Certain regions of the $\lambda$ genome are not required for infectivity under lab conditions.
- These regions can be deleted and replaced with foreign DNA.
- This forms the basis of insertion vectors and replacement vectors derived from $\lambda$.
- Regulatory region:
- Controls the decision between lytic and lysogenic cycles.
- Elements from this region (promoters and operator sites) have become standard regulatory parts in cloning and expression systems.
Phage-Based Cloning Vectors
Phage-derived vectors complement plasmid vectors by allowing larger DNA inserts and efficient DNA delivery into bacteria.
Lambda Insertion and Replacement Vectors
- Insertion vectors:
- A small non-essential region in $\lambda$ is replaced with a multiple cloning site (MCS).
- Suitable for relatively modest-sized inserts.
- Used to construct phage libraries or to express small genes.
- Replacement vectors:
- A larger non-essential fragment of $\lambda$ DNA (the “stuffer fragment”) is removed.
- The removed segment is replaced by foreign DNA of comparable size.
- Can carry inserts typically up to ~20 kb or more.
- Useful for genomic libraries where large genomic fragments are needed.
Advantages of $\lambda$ vectors:
- High infection efficiency compared with transformation of plasmids.
- Easy screening via plaques.
- Ability to carry larger DNA fragments than many standard plasmids.
Cosmids
Cosmids are hybrid vectors that combine:
- A plasmid backbone (with origin of replication, selectable marker),
- And cos sites from phage $\lambda$.
Key features:
- Can accept very large inserts (often 35–45 kb).
- Because of the cos sites, cosmid DNA can be packaged into phage heads in vitro, then introduced into bacteria by infection, which is more efficient than transformation.
- Once inside the bacterium, the cosmid behaves like a plasmid, replicating autonomously.
Uses:
- Construction of genomic libraries of higher organisms.
- Cloning of fragments too large for standard plasmids.
Bacteriophages in DNA Library Construction and Screening
Phage-based vectors have historically been important in constructing and analyzing DNA libraries.
Genomic Libraries in Phage Vectors
- Genomic DNA is fragmented (often partially, to preserve large overlapping fragments).
- Fragments of suitable size are ligated into $\lambda$ replacement vectors or cosmids.
- The recombinant DNA is packaged into phage heads in vitro.
- Bacteria are infected; each infected bacterium produces a plaque corresponding to one library clone.
Advantages:
- Even coverage of the genome when sufficiently many independent clones are made.
- Plaque lifts: DNA from plaques can be transferred to membranes and hybridized with labeled probes to identify clones containing sequences of interest (hybridization techniques are treated elsewhere).
cDNA Libraries in Phage Vectors
Although often associated with plasmids, cDNA libraries (DNA copies of mRNA populations) have also been constructed in phage vectors:
- Especially in phage display systems (see below) or in specialized $\lambda$ expression vectors.
- The vector is designed so that the cDNA is placed downstream of a phage promoter and sometimes fused to a phage protein, allowing expression and/or display.
Transduction: Natural Phage-Mediated Gene Transfer
Phages can naturally move DNA between bacteria, a process called transduction.
There are two main types relevant to genetic engineering concepts:
Generalized Transduction
- Occurs mainly with some lytic or virulent phages (e.g., P1, P22).
- During phage assembly, by mistake, fragments of host bacterial DNA are packaged instead of phage DNA.
- When such a transducing particle infects a new bacterium, it introduces pure bacterial DNA.
- This DNA can recombine with the recipient’s chromosome.
Implications:
- Demonstrates horizontal gene transfer via phages in nature.
- In the lab, generalized transduction is used for fine-scale genetic mapping and for moving genes or mutations between bacterial strains.
Specialized Transduction
- Associated with temperate phages (e.g., $\lambda$) that integrate at specific sites in the bacterial chromosome.
- When the prophage excises incorrectly, it may carry along exactly defined adjacent bacterial genes.
- Only these specific regions can be transduced, hence “specialized.”
Implications:
- Provides a very precise method of moving defined bacterial genes.
- Helped reveal the organization and regulation of bacterial genomes.
- Conceptually important for understanding how bacterial pathogens can acquire virulence factors via phages.
Phage Display (Conceptual Overview)
While detailed methods of phage display belong under applications and specific techniques, its basic principle is closely tied to phage biology:
- DNA encoding a peptide or protein variant is fused to a gene for a phage coat protein.
- The resulting phage displays the peptide/protein on its surface.
- Large libraries of different variants can be screened by binding to a target molecule.
- Bound phage are amplified, linking genotype (DNA inside the phage) and phenotype (displayed protein).
This method relies on:
- The efficient production of phage particles.
- The stability of phage coat proteins even when fused to foreign peptides.
- The ease of selection and amplification via infection and plaque formation.
Safety and Practical Considerations
In genetic engineering labs, bacteriophages are typically handled under appropriate biosafety levels, because:
- Some phages can carry bacterial toxin genes or antibiotic resistance genes in nature.
- Phages can rapidly lyse bacterial cultures, which is desirable in some experiments but problematic in others.
Engineered phage vectors are usually:
- Attenuated or disarmed (key genes deleted or disabled),
- Designed to be host-range restricted, infecting only specific lab strains,
- And constructed to minimize unintended gene transfer.
These modifications make phage-based genetic engineering controllable and predictable in the laboratory setting.
Summary: Why Bacteriophages Matter in Genetic Engineering
- They are natural vehicles for DNA delivery into bacteria.
- Temperate phages like $\lambda$ provide integrative and regulatory systems useful for stable gene insertion.
- Phage-derived vectors (including $\lambda$ vectors and cosmids) enable cloning of larger DNA fragments than standard plasmids and support efficient DNA library construction.
- Phage-mediated transduction explains and exploits horizontal gene transfer.
- Techniques such as phage display use phage biology to link DNA sequences to protein functions, allowing powerful selection and screening.
Understanding bacteriophages as genetic engineering tools provides a foundation for many classical and modern molecular methods that exploit their unique life cycles and structures.