VECTOR BIOLOGY: CLONING AND EXPRESSION VECTORS

GROUP 3

OUTLINE
Introduction Definition of terms Essential features of cloning and expression vectors Types of cloning vectors Plasmids Virus-based vectors ( phage, M13 phage) Hybrid vectors (Cosmids, phagemids) Artificial chromosomes Cloning vectors in animals Cloning vectors in higher plants Expression vectors

Introduction
Many recombinant DNA methods require numerous copies of a specific DNA fragment. gene cloning provides a means of generating identical copies (clones) of an original piece of DNA. It involves separating a specific gene or DNA segment from a larger chromosome, attaching it to a small molecule of carrier DNA (vector), and then replicating this modified DNA thousands or millions of times through both an increase in cell number and the creation of multiple copies of the cloned DNA in each cell. The result is selective amplification of a particular gene or DNA segment.

Definition of terms
A clone is a population of identical cells, generally those containing identical recombinant DNA molecules. A vector (in molecular biology) is a DNA molecule capable of replication in a host organism, which serves as a carrier for a foreign DNA molecule A cloning vector is a stable, self-replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell in order to generate multiple identical copies of the foreign DNA. An expression vector is a cloning vector designed so that a foreign gene inserted into the vector will be expressed in the host organism. Host cell in which the recombinant DNA is introduced, for which the vector has replication functions- bacteria, yeast, mammalian cells, etc

Essential features of Cloning vectors

An effective cloning vector has three important characteristics: An origin of replication, which ensures that the vector is replicated within the cell; Selectable markers, which enable any cells containing the vector to be selected or identified. One or more unique restriction sites into which a DNA fragment can be inserted.

Major types of cloning vectors

There are four major types of cloning vectors applied for cloning in bacteria and yeast host cells: 1. Plasmids

2. Virus-based vectors (Bacteriophages ) 3. Hybrid vectors (Cosmids, phagemids) 4. Artificial chromosomes (Yeast and Bacterial artificial chromosomes).

PLASMIDS
extra-chromosomal elements of DNA, which are relatively small, covalently closed circular molecules carrying genes for antibiotic resistance, conjugation, virulence or the metabolism of unusual substrates. Plasmids lead an independent existence in the bacterial cell. pBR322 one of the most notable plasmids named after its developers Bolivar and Rodriguez in the early 1970s.

Features
At least one DNA sequence that can act as an origin of replication. Possess at least one unique restriction site. antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain plasmid. Range in size from about 1.0kb for the smallest to over 250kb for the largest plasmids May exhibit stringent or relaxed regulation of replication Insert capacity: approx 10-20kb. A few types of plasmid are also able to replicate by inserting themselves into the bacterial chromosome. These integrative plasmids - episomes

Classification of plasmids
Fertility or F plasmids: carry genes (tra genes) that possess the ability to promote sexual conjugation between bacterial cells. Resistance or R plasmids: carry genes conferring on the host bacterium resistance to one or more antibacterial agents Col plasmids: these contains genes that code for colicins i. e. proteins that kill other bacteria. An example is ColE1 of Escherichia coli. Degradative plasmids: these allow the host bacterium to metabolize unusual molecules e.g. toluene and salicylic acid. Virulence plasmids: these confer pathogenicity on the host bacterium.

Cloning with pBR322

pUC plasmids
Allows for identification of the foreign DNA containing cells in a single screening step. Have the advantage of a High copy number, A multiple cloning site or polylinker. pUC8, for example, is a Lac-selection plasmid. This plasmid carries the ampicillin resistance gene and a gene called lac Z, which codes for part of the enzyme beta-galactosidase. Cloning with pUC8 involves insertional inactivation of the lac Z gene, with recombinants identified because of their inability to synthesize the amino terminal portion of -galactosidase called the peptide.

Cloning with pUC8

Cells that harbor a normal pUC8 plasmid are ampicillin resistant and able to synthesize beta-galactosidase; recombinants are also ampicillin resistant but are unable to make beta-galactosidase. Screening for beta-galactosidase presence or absence is done using a lactose analogue called X-gal (5- bromo-4chloro-3 indoyl--D-galactopyranoside) which is broken down by beta-galactosidase to a product that is colored deep blue. If X-gal is added to the agar, along with ampicillin, then non-recombinant colonies, the cells of which synthesize beta-galactosidase will be colored blue, whereas recombinants with a disrupted lac Z gene and unable to synthesize beta-galactosidase will be white.

Bacteriophage
a typical example of a head-and-tail phage DNA molecule is 49 kb in size. is a linear, double-stranded DNA phage and consists of two self-complementary 12bp single-stranded tail at both ends, called cohesive termini (COS SITES). Being complementary, they can base-pair with one another to form a circular, completely double-stranded molecule, a necessary prerequisite for insertion into the bacterial genome. It selectively infects bacteria and replicates by a lytic or non-lytic (lysogenic) pathway.

 Two basic types of vectors: 1. insertional vectors 2. replacement vectors

M13 phage vectors

M13 is an example of a filamentous phage. It is also a male-specific lysogenic phage Its DNA molecule is much smaller than the genome ((about 6407 nucleotides in length). It is circular and is unusual in that it consists entirely of singlestranded DNA. Because M13 switches between a single-stranded form and a double-stranded replicative form it is of utmost importance in in vitro mutagenesis experiments. normal M13 genome is 6.4kb in length, but most of this is taken up by ten closely packed genes, each essential for the replication of the phage. There is only a single, 507 nucleotide inter-genic sequence into which new DNA could be inserted. Inserts larger than a few fragments tend to make the vector unstable.

Cosmid vectors
hybrids between a phage DNA molecule and a bacterial plasmid. two cos sites from phage that are separated by 3751 kbp of intervening sequence. Cosmids can be isolated as dsDNA for in vitro manipulation.

Phagemids
Phagemids are plasmids that contain the f1 phage origin of replication for the production of single stranded DNA. They are small plasmids and have the ability to accept larger inserts than M13-based vectors Phagemids can only produce ssDNA in the presence of a wild-type M13 or f1 helper phage. In the absence of a helper phage, dsDNA can be isolated as a normal plasmid

phagemids

Artificial Chromosoomes

ARTIFICIAL CHROMOSOMES
Bacterial artificial chromosomes (BACs), mammalian artificial chromosomes(MACs) viral PI artificial chromosomes (PACs) Yeast artificial chromosomes (YACs).

EXPRESSION VECTORS
Especially useful in the production of large amounts of a low-abundance protein e.g. many protein hormones and other signaling or regulatory proteins are normally expressed at very low concentrations, precluding their isolation and purification in large quantities by standard biochemical techniques. insulin, growth hormone, factor VIII (a blood clotting factor), granulocyte colony-stimulating factor (G-CSF) and other human proteins with therapeutic uses.

 Expression vectors, in bacteria, in addition to the usual origin of replication, restriction sites, and selectable markers, contains sequences required for transcription and translation in bacterial cells. These additional sequences may include: 1. A bacterial promoter, such as the lac promoter. 2. A DNA sequence that, when transcribed into RNA, produces a prokaryotic ribosome binding site. 3. Prokaryotic transcription initiation and termination sequences. 4. Sequences that control transcription initiation, such as regulator genes and operators

The general layout of expression vectors