UNIT 2 NOTES — VECTORS

Subject: rDNA Technology (BBT0405)

Detailed easy-language notes for 10-mark answers.

1. Introduction to Vectors

Definition

A vector is a DNA molecule used to carry foreign DNA into a host cell where it can replicate
and express.

Vectors are important tools in recombinant DNA technology.

2. Types of Vectors

Main types:

1. Cloning vectors

2. Expression vectors

3. Promoterless vectors

4. Plasmids

5. Bacteriophage vectors

6. Cosmids

7. Phagemids

8. BACs

9. YACs

10. Shuttle vectors

3. Cloning Vectors

Definition
Cloning vectors are DNA molecules used to produce multiple copies of foreign DNA inside
host cells.

Examples

 pBR322

 pUC18

 Lambda phage

 Cosmids

Features of Ideal Cloning Vector

1. Origin of Replication (ori)

Starts replication inside host.

2. Selectable Marker

Helps identify transformed cells.

3. Multiple Cloning Site (MCS)

Contains restriction enzyme sites.

4. Small Size

Easy handling and transfer.

5. High Copy Number

Produces many copies.

4. Expression Vectors

Definition

Expression vectors are vectors specially designed for protein production.

Components
  Strong promoter

 Ribosome binding site

 Terminator

 Marker gene

 MCS

Importance

Helps:

 Transcription

 Translation

 High-level protein synthesis

Applications

 Insulin production

 Vaccine production

 Industrial enzymes

5. Promoterless Vectors

Definition

Vectors lacking promoter sequence are called promoterless vectors.

Uses

 Promoter study

 Gene regulation analysis

 Reporter gene studies

Definition

Plasmids are small circular extra-chromosomal DNA molecules found in bacteria.

They replicate independently.

Features

 Circular DNA

 ori present

 Selectable marker

 MCS

Applications

 Gene cloning

 Protein production

 DNA transfer

7. Selectable Marker Gene

Definition

A gene used to identify transformed cells.

Common Marker Genes

Marker Resistance

ampR Ampicillin

tetR Tetracycline

kanR Kanamycin

cmR Chloramphenicol
Marker Resistance

Importance

Only transformed cells survive on antibiotic media.

8. pBR322 Vector

Definition

pBR322 is one of the earliest plasmid vectors used in genetic engineering.

Features

 Size = 4363 bp

 ampR gene

 tetR gene

 Multiple restriction sites

Important Restriction Sites

 BamHI

 HindIII

 EcoRI

Screening of Recombinants

If foreign DNA inserted into tetR:

 tetR becomes inactive

 Recombinant cells become tetracycline sensitive

Selection:
  Growth on ampicillin

 No growth on tetracycline

9. pUC18 / pUC19 Vectors

Features

 Small size (~2.7 kb)

 High copy number

 ampR gene

 lacZ gene

 MCS inside lacZ

Importance

Used in blue-white screening.

10. Blue-White Screening

Principle

Distinguishes recombinant and non-recombinant colonies.

Mechanism

Non-Recombinant

 lacZ active

 β-galactosidase formed

 Blue colonies

Recombinant

 lacZ disrupted

 No enzyme
  White colonies

Result

Colony Meaning

Blue Non-recombinant

White Recombinant

11. Lac Operon

Components

 lacI

 lacZ

 lacY

 lacA

Functions

lacZ

β-galactosidase production.

lacY

Permease production.

lacA

Transacetylase production.

Importance

Used in cloning and screening.

12. Bacteriophages
Definition

Viruses that infect bacteria.

Structure

 Head

 Tail

 Tail fibers

Importance

Used as cloning vectors.

13. Lambda (λ) Phage Vectors

Definition

Vectors derived from bacteriophage lambda.

Features

 Large insert capacity

 Efficient infection

 Easy screening

Insert Capacity

9–23 kb

14. Types of Lambda Vectors

1. Insertion Vectors

Features
  DNA inserted at one restriction site

 Small insert size

Example

 λgt10

2. Replacement Vectors

Features

 Part of vector DNA replaced

 Large insert capacity

Examples

 λEMBL3

 λEMBL4

15. Advantages of Lambda Vectors

1. High efficiency

2. Large DNA cloning

3. Easy plaque screening

4. Stable cloning

5. Rapid replication

16. Lytic Cycle

Definition

Virus multiplies and destroys host cell.

Steps

1. Attachment
 2. Penetration

3. Biosynthesis

4. Assembly

5. Lysis

Result

New phages released.

17. Lysogenic Cycle

Definition

Phage DNA integrates into bacterial chromosome.

Steps

1. Attachment

2. Injection

3. Integration

4. Replication with host

5. Induction

Importance

Host cell survives.

18. Phagemids

Definition

Hybrid vectors containing plasmid and filamentous phage properties.

 Plasmid ori

 f1 ori

 Marker gene

 MCS

Uses

 ssDNA production

 Sequencing

 Phage display

19. Cosmid Vectors

Definition

Hybrid vectors containing:

 Plasmid DNA

 Lambda cos sites

Features

 Insert size 35–45 kb

 ori present

 Marker gene present

Applications

 Genomic libraries

 Large DNA cloning

Definition

BACs are large cloning vectors derived from E. coli F-plasmid.

Features

 Insert size = 100–300 kb

 High stability

 Low copy number

Components

 oriS

 repE

 parA/parB

 CmR gene

Applications

 Genome sequencing

 Human genome project

21. YAC (Yeast Artificial Chromosome)

Definition

Artificial chromosome vectors used in yeast.

Features

 Insert size up to 1–2 Mb

 Replicate like chromosome

Component Function

ARS Replication

CEN Segregation

TEL Stability

URA3/TRP1 Selection

Applications

 Genome mapping

 Large DNA cloning

22. Difference Between BAC and YAC

Feature BAC YAC

Host E. coli Yeast

Insert size 300 kb 1–2 Mb

Stability High Lower

23. Animal Viral Vectors

Definition

Modified animal viruses used for gene transfer.

Types

1. Retrovirus

2. Adenovirus

3. Herpes virus
Applications

 Gene therapy

 Protein production

24. Plant Viral Vectors

Definition

Plant viruses used for foreign gene delivery into plants.

Examples

 TMV

 PVX

 CPMV

 Geminivirus

Uses

 Vaccine production

 Plant biotechnology

25. Baculovirus Vector System

Definition

Expression system using insect viruses for protein production.

Features

 Infect insect cells

 High protein expression

Important Promoters

 Polyhedrin promoter

 p10 promoter

Steps

1. Gene insertion

2. Co-transfection

3. Homologous recombination

4. Recombinant baculovirus formation

5. Protein production

Applications

 Recombinant vaccines

 Therapeutic proteins

26. Pichia Vector System

Definition

Yeast-based expression system using Pichia pastoris.

Advantages

 High protein yield

 Eukaryotic modifications

 Fast growth
Applications

 Enzyme production

 Recombinant proteins

27. Ti Plasmid Vector

Definition

Plasmid of Agrobacterium tumefaciens used for plant transformation.

Features

 Large plasmid (~200 kb)

 Transfers T-DNA into plants

Important Regions

Region Function

T-DNA Gene transfer

vir genes DNA transfer

Border sequences Define T-DNA

Uses

 Transgenic plants

 Plant genetic engineering

28. Ri Plasmid Vector

Definition

Plasmid of Agrobacterium rhizogenes causing hairy root disease.

 rol genes

 vir genes

 T-DNA

Uses

 Root culture

 Secondary metabolite production

29. Shuttle Vectors

Definition

Vectors capable of replication in two different hosts.

Example:

 E. coli and yeast

Features

 Two ori

 Two marker genes

 MCS

Applications

 Gene transfer

 Cloning in multiple hosts

30. Difference Between Plasmid and Bacteriophage

Nature Circular DNA Viral DNA

Insert size Small Large

Transfer Transformation Infection

31. Difference Between Insertion and Replacement Vectors

Feature Insertion Vector Replacement Vector

Mechanism DNA inserted DNA replaced

Insert size Smaller Larger

Example λgt10 λEMBL3

32. Difference Between Phagemid and Cosmid

Feature Phagemid Cosmid

Hybrid type Plasmid + M13 Plasmid + λ

Insert size Small Large

Main use ssDNA production Genomic library

Important PYQ Topics

1. Features of cloning vectors

2. pBR322 vector

3. Blue-white screening

4. Lambda phage vectors

5. Lytic and lysogenic cycle

6. Cosmids and phagemids

7. BAC and YAC

9. Shuttle vectors

10. Expression vectors

11. Difference between insertion and replacement vectors

12. Animal and plant viral vectors