Bacteriophage Lambda and Cloning Vectors

Bacteriophage Lambda

• Bacteriophage lambda is a bacterial virus that infects Escherichia coli.
• Its structure includes a head, tail, and tail fibers.
• Viral DNA is linear, double-stranded, and 48.5 kbp with 12 base pair sticky ends.
• The sticky ends are complementary, allowing them to hybridize.

Bacteriophage Lambda as a Cloning Vector

• Lambda phage is used as a vector using two strategies.
• Insertional vectors: The insert DNA is cloned into the multiple cloning site (MCS) within the lacZ coding sequence. Cloning capacity is 13 kbp.
• Replacement vectors (or substitution vectors): A central stuffer is swapped with the insert DNA. Cloning capacity is 23 kbp.
• Substitution vectors are generally preferred but more complex to implement.

Cosmid as a Cloning Vector

• Cosmids are artificial vectors, essentially plasmids with one or two cos sites from lambda phage.
• The cos sites allow in vitro packaging of cosmid DNA into lambda phage particles.
• Insert size can be up to 45 kbp.
• Cosmids contain plasmid features (origin of replication, multiple cloning site, selectable marker) and lambda phage features (cos sites).

Advantages and Limitations of Cosmids

• Advantages: Cloning of large inserts (up to 45 kbp), infection process (rather than transformation), maintenance of recombinant phage particles, and cosmid reproduction as a large plasmid without destroying the infected bacteria.
• Limitations: Complex realization and DNA rearrangements can occur.

Agrobacterium as a Vector

• Agrobacterium tumefaciens is a gram-negative soil bacterium that naturally transforms plant cells, resulting in crown gall tumors.
• Infection occurs through breaks or wounds.
• Tumor formation results from T-DNA integration into the plant genome.

Ti Plasmid of Agrobacterium

• Virulent strains of A. tumefaciens harbor large plasmids (140-235 kbp) often called Ti plasmids, with elements like:
  • T-DNA (with left and right borders).
  • Virulence (vir) region genes involved in T-DNA transfer.
  • Origin of replication.
  • Region enabling conjugative transfer.
  • O-cat region (for opine catabolism).

Ti Plasmid Based Vectors

• Binary systems: Uses two vectors: a disarmed Ti plasmid (with gene of interest but no vir genes), and a helper vector (with vir genes).
• Co-integrated vectors: Involves three vectors; a disarmed Ti plasmid, an intermediate vector, and the helper vector.

Retroviral Cloning Vectors

• Retroviral vectors are capable of reverse-transcribing RNA into DNA, enabling stable integration into the host genome.
• Useful for introducing foreign genes into target cells.
• Widely used vectors (based on RNA viruses) include murine leukemia viruses and lentiviruses.

Retroviral Vector Genome

• Contains:
  • LTR (long terminal repeat)
  • PBS (primer binding site)
  • PPT (polypurine track)
  • "pol" gene
  • "gag" gene
  • "env" gene
  • Packaging signal site
  • 5' and 3' untranslated regions

Shuttle Cloning Vectors

• Shuttle vectors can replicate in two different organisms (e.g., bacteria and yeast).
• They include two origins of replication and two selection markers, one for each organism.

Artificial Chromosome Cloning Vectors

• Include Yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs).
• Useful for genome sequencing, functional characterization of genomic regions, and transduction of large DNA segments.

YACs

• Genetically engineered yeast chromosomes.
• Consist of two telomeric sequences, a yeast centromere, a yeast ARS, and selectable markers (e.g., URA3).
• Average cloning capacity of 100 kbp to 2 Mbp.

BACs

• Designed for cloning large DNA fragments (100-300 kbp) in E. coli.
• Contain a single copy F-plasmid origin of replication.
• Useful for carrying various genetic material.

P1-derived Artificial Chromosomes (PAC)

• Derived from the DNA of P1 bacteriophages, and bacterial artificial chromosome.
• Can carry larger amounts (100-300 kb) of DNA.

Human Artificial Chromosomes (HAC)

• Mini-chromosomes constructed artificially in human cells.
• Have their own self-replicating and segregating systems, behaving independently from host chromosomes.

Expression Vectors

• Plasmids or viruses designed for expressing genes (to produce protein).
• Have elements like origin of replication, multiple cloning site (MCS), regulatory elements (promoters, enhancers, terminators, initiation sites, stop codons), and selectable markers.

Methods for Direct DNA Transfer into Eukaryotic Cells (non-viral)

• Calcium Phosphate: DNA forms a precipitate taken up by the cells via endocytosis.
• Electroporation: High voltage electrical pulses create transient pores in the plasma membrane, allowing DNA entry.
• Microinjection: DNA is directly injected into cells using a fine glass needle/micropipette.
• Liposomes: Hollow spheres of phospholipid that carry DNA into cells.
• Gene Gun: Propel DNA-coated particles into cells; useful for plant tissues.

Nucleic Acid Extraction

• Isolates DNA from cellular components for further investigations (PCR, sequencing, etc.). 
• Two main methods:
  • Phenol-chloroform extraction
  • Solid-phase extraction

Gel Electrophoresis

• Separates DNA fragments based on size.
• Agarose is a linear polymer gel used.

Molecular Hybridization

• Association of complementary single-stranded nucleic acids.
• Hydrogen bonds (A-T, C-G) form a double-strand or duplex.
• Probes are used for identifying specific nucleic acid sequences. They can either use radioactive or non-radioactive tags.