Unit–2: Cloning and Expression Vectors (Detailed Notes)
Primary References: T. A. Brown – Gene Cloning and DNA Analysis; Smita Rastogi & Neelam Pathak – Genetic
Engineering
Prepared for: [Link]. Microbiology (University Examination + CSIR/UGC NET)
1. Vectors in Recombinant DNA Technology
A vector is a DNA molecule capable of autonomous replication inside a host cell and used to carry a foreign DNA
fragment (insert) into the host.
Basic Properties of an Ideal Vector
• Origin of replication (ori)
• Selectable marker genes
• Unique restriction sites (MCS)
• Small size and high copy number
• Stable maintenance inside host
2. Plasmid Vectors
Plasmids are extrachromosomal, circular, double-stranded DNA molecules found mainly in bacteria.
Commonly Used Plasmid Vectors
• pBR322 – antibiotic resistance markers (ampR, tetR)
• pUC series – high copy number, lacZ based screening
• pBluescript – MCS and blue-white screening
Cloning Strategy Using Plasmids
• Restriction digestion of vector and insert
• Ligation to form recombinant plasmid
• Transformation into E. coli
• Selection and screening of recombinants
3. Bacteriophage Vectors
Bacteriophages are viruses that infect bacteria and can be used as cloning vectors for larger DNA fragments
compared to plasmids.
λ (Lambda) Phage Vector
• Insertion vectors – insert size up to ~7 kb
• Replacement vectors – insert size up to ~20 kb
• Packaging requirement between 38–52 kb
M13 Phage Vector
• Single-stranded DNA production
• Useful for sequencing and mutagenesis
4. Chimeric Vectors
Chimeric vectors combine features of different vectors to improve cloning efficiency.
• Cosmids – plasmid + cos sites of λ phage (insert up to 45 kb)
• Phagemids – plasmid + phage origin
5. Artificial Chromosome Vectors
Yeast Artificial Chromosomes (YAC)
• Contain telomeres, centromere, and ARS
• Insert size: up to 1 Mb
• Used for cloning large eukaryotic DNA
Bacterial Artificial Chromosomes (BAC)
• Based on F-plasmid
• Insert size: 100–300 kb
• High stability, used in genome projects
Human/Mammalian Artificial Chromosomes (HAC/MAC)
• Autonomously replicating chromosomes
• Used in gene therapy and functional genomics
6. Viral Vectors for Plant and Animal Cells
• Plant viral vectors: CaMV, TMV
• Animal viral vectors: Adenovirus, Retrovirus, Lentivirus
• High transfection efficiency
• Used in gene delivery and gene therapy
7. Expression Vectors
Expression vectors are designed to allow transcription and translation of the cloned gene.
Essential Components
• Strong promoter
• Ribosome binding site (RBS)
• Terminator sequence
• Selectable marker
Expression Systems
• E. coli – fast growth, simple regulation
• Yeast – post-translational modification
• Insect cells – baculovirus system
• Mammalian cells – authentic protein processing
8. Shuttle Vectors and Gene Trapping Vectors
• Shuttle vectors replicate in two different hosts
• Contain two ori and selectable markers
• Gene trapping vectors used to identify unknown genes
9. Vector Design and Modification Strategies
• Codon optimization
• Fusion tags (His-tag, GST)
• Signal peptides for secretion
• Promoter engineering
10. Chemical Synthesis of Oligonucleotides
Oligonucleotides are chemically synthesized short DNA sequences used as primers, probes, and adapters.
• Solid-phase phosphoramidite method
• Synthesis in 3′ → 5′ direction
• Automated DNA synthesizers
11. Exam-Oriented Notes (University + NET)
• Draw vector maps wherever possible
• Compare plasmid vs phage vs BAC/YAC
• NET focus: insert size limits and expression systems
• Use examples (pBR322, λ phage, BAC) in answers