CLONING VECTORS AND GENE CONSTRUCTS

SUBMITTED TO:Dr. Shyamalamma.SProfessorDepartment of Plant BiotechnologyGKVK, UAS Bangalore

SUBMITTED BY:K.KavyaID NO.:- PALB 5269Jr.M.Sc.(Agri.) Plant BiotechnologyGKVK,UAS Bangalore

CONTENTS

Introduction Vector Simple & expression vector Purpose of the expression vector Steps in molecular cloning Types of vectors Desirable features of a plasmid vector Features of artificially constructed vector Screening procedures DNA construct Gene cassette and integrons

INTRODUCTIONMolecular cloning, has spurred progress

throughout the life sciences. Beginning in the 1970s, with the discovery of restriction endonucleases recombinant DNA technology has seen exponential growth in both application and sophistication, yielding increasingly powerful tools for DNA manipulation.

Cloning genes is now so simple and efficient that it has become a standard laboratory technique. This has led to an explosion in the understanding of gene function in recent decades. Emerging technologies promise even greater possibilities, such as enabling researchers to seamlessly stitch together multiple DNA fragments and transform the resulting plasmids into bacteria, in under two hours, or the use of swappable gene cassettes, which can be easily moved between different constructs, to maximize speed and flexibility.

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VECTOR• In molecular cloning, a vector is a DNA

molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA.

HISTORY OF CLONING

RODRIGUEZ RAYMON

Rodriguez Raymon along with Paco Bolivar constructed the vector “pBR 322” in the year 1977.

Every engineered vector consists of the following:• Origin of replication• Multi-cloning site • Selectable marker

SIMPLE AND EXPRESSION VECTORS• SIMPLE VECTOR:- Simple vectors called

transcription vectors are only capable of being transcribed but not translated : they can be replicated in a target cell but not expressed.

• EXPRESSION VECTORS :- Transcription vectors are used to not only amplify their insert but also express the gene in an organism.

The expression vector generally has a promoter sequence that drives the expression of the transgene.

PURPOSE OF THE EXPRESSION VECTOR

Gene Isolation

Step 1

Multiplication

Step 2

Gene expression

Step 3

• The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.

• The process of insertion of the vector : (a) Transformation in bacterial cells.

(b) Transfection in eukaryotic cells.

But if a viral vector is inserted, it is called

transduction.

CLONING VECTORA cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

Cloning is generally first performed using Escherichia coli,

Some vectors also include elements that allow them to be maintained in another organism in addition to E. coli, and these vectors are called shuttle vector.

STEPS IN MOLECULAR CLONING

A• 1. Choice of host organism and cloning vector• 2. Preparation of vector DNA and DNA to be cloned.

B• 3. Creation of recombinant DNA• 4. Introduction of recombinant DNA into host organism

C• 5. Selection of organisms containing recombinant DNA• 6. Screening for clones with desired DNA inserts and biological properties

TYPES OF VECTORS The four major types of vectors are 1. Plasmids, 2. Bacteriophages (as phage λ)

3. Cosmids, and 4. Artificial chromosomes.

PLASMID Autonomously replicating circular extra-chromosomal DNA.

They are the standard cloning vectors and the most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb

in size.

The bacteriophages used for cloning are the phage λ and M13 phage.Some plasmids contain an M13 bacteriophage origin of replication and may be used to generate single-stranded DNA. These are called phagemid, and examples are the pBluescript series of cloning vectors.

BACTERIOPHAGE

There is an upper limit on the amount of DNA that can be packed into a phage

(a maximum of 53 kb), therefore to allow foreign DNA to be inserted into phage DNA, phage cloning vectors need to have some non-essential genes deleted, for e.g., the genes for lysogeny in phage λ. 

There are two kinds of λ phage vectors - insertion vector and replacement vector.

Insertion vectors contain a unique cleavage site whereby foreign DNA with size of 5–11 kb may be inserted.

In replacement vectors, the cleavage sites flank a region containing genes not essential for the lytic cycle, and this region may be deleted and replaced by the DNA insert in the cloning process, and a larger sized DNA of 8–24 kb may be inserted.

COSMIDCosmids are plasmids that incorporate a segment of bacteriophage λ DNA that has the cohesive end site (cos) which contains elements required for packaging DNA into λ particles. It is normally used to clone large DNA fragments between 28 to 45 Kb.

Bacterial artificial chromosomeInsert size of up to 350 kb can be cloned in bacterial artificial chromosome (BAC). BACs are maintained in E. coli with a copy number of only 1 per cell.

Yeast artificial chromosome

They are very useful in mapping human genome as they could accommodate hundreds of thousands of bp.

Insert of up to 3,000 kb may be carried by YAC.

Human artificial chromosome Human artificial chromosome may be potentially useful as a gene transfer

vectors for gene delivery into human cells, and a tool for expression studies and determining human chromosome function.

It can carry very large DNA fragment (there is no upper limit on size for practical purposes), therefore it does not have the problem of limited cloning capacity of other vectors, and it also avoids possible insertional mutagenesis caused by integration into host chromosomes by viral vector.

DESIRABLE FEATURES OF ANY PLASMID

VECTOR1. It should be of a small size.2. It should Confer a selectable phenotype on the host cells

so that the transformed cells can be selected for.3. Should contain single sites for a large number of

restriction enzymes to enable efficient production of recombinant vectors.

4. Should enable the identification of bacterial colonies containing recombinant plasmids.

FEATURES OF AN ARTIFICIALLY CONSTRUCTED VECTOR

1. Origin of replication2. Promoter3. Cloning site/Multiple cloning site4. Selectable marker/Genetic markers5. Antibiotic resistance6. Epitope7. Reporter genes8. Protein purification tags

1. ORIGIN OF REPLICATION: Necessary for the replication and maintenance of the vector in the host cell.2. PROMOTER: Promoters are used to drive the transcription of the vector's transgene as well as the other genes in the vector such as the antibiotic resistance gene. 3. CLONING SITE: This may be a multiple cloning site or other features that allow for the insertion of foreign DNA into the vector through ligation.

4. GENETIC MARKERS: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.6. EPITOPE: Vector contains a sequence for a specific epitope that is incorporated into the expressed protein. Allows for antibody identification of cells expressing the target protein.

7. REPORTER GENES: Some vectors may contain a reporter gene that allow for identification of plasmid that contains inserted DNA sequence. An example is lacZ-α which codes for the N-terminus fragment of β-galactosidase, an enzyme that digests galactose. Green Fluorescent Protein (GFP):- It is rapidly becoming a widely used used reporter gene. It is easier to assay than GUS and is a non-destructive method. GFP can be used in situations where GUS cannot, for e.g., In screening primary transformants, in time-course experiments, or for analyzing segregation in small seedlings.

The GFP is actually located in discrete spots around the bell margin of the jellyfish and will fluoresce under certain conditions.

To work efficiently in plants “gfp gene” has to be modified significantly in order to :1. Remove a cryptic intron2. Make the codon usage more ‘plant-like’3. Prevent accumulation in the nucleoplasm.The mutant form of GFP used in pGREEN makes the bacteria a yellow-green color even in white light.

Jelly fish Aequorea victoria

8. PROTEIN PURIFICATION TAGS: Some expression vectors include proteins or peptide sequences that allows for easier purification of the expressed protein. Examples include polyhistidine-tag, glutathione-S-transferase, and maltose binding protein. Some of these tags may also allow for increased solubility of the target protein.

SCREENING PROCEDURE OF CLONING VECTORS

• The most widely used system for detecting the presence of a cloned DNA fragment is the gene coding for E. coli β-galactosidase, whose activity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'-dichloro indigo).

(A) BLUE/WHITE SELECTION

(B) REPLICA PLATING TECHNIQUEReplica plating is a microbiological technique in which one or more secondary Petri plates containing different solid (agar-based) selective growth media (lacking nutrients or containing chemical growth inhibitors such as antibiotics) are inoculated with the same colonies of microorganisms from a primary plate (or master dish), reproducing the original spatial pattern of colonies.

The purpose of replica plating is to be able to compare the master plate and any secondary plates, typically to screen for a desired phenotype.

It is more correct to refer to "negative screening”.

(B) REPLICA PLATING TECHNIQUE

The development of replica plating required two steps.

1. The first step was to define the problem: a method of identifiably duplicating colonies.

2. The second step was to devise a means to reliably implement the first step.

Esther Lederberg and Joshua Lederberg

DNA CONSTRUCTA DNA construct is an artificially constructed segment of nucleic acid that is going to be "transplanted" into a target tissue or cell. It often contains a DNA insert, bacterial resistance genes and promoters. A DNA construct may express wildtype protein, prevent the expression of certain genes by expressing competitors or inhibitors, or express mutant proteins, such as deletion mutations or missense mutations. A DNA construct is often used in molecular biology to analyze macromolecules such as proteins or RNA in more detail.

GENE CASSETTE & INTEGRONS

A GENE CASSETTE is a type of mobile genetic element that contains a gene and a recombination site.

They may exist as incorporated into an integron or freely as circular DNA. Gene cassettes often carry antibiotic resistance genes.

An example would be the kanMX cassette which confers kanamycin (an antibiotic) resistance upon bacteria.

INTEGRONS are genetic structures in bacteria which express and are capable of acquiring and exchanging gene cassettes.

These cassettes typically carry a single gene without a promoter.

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In genetic engineering, a gene cassette refers to a manipulable fragment of DNA carrying, and capable of expressing, one or more genes of interest between one or more sets of restriction sites. It can be transferred from one DNA sequence (usually on a vector) to another by 'cutting' the fragment out using restriction enzymes and 'pasting' it back into the new context

APPLICATIONS A particular gene can be isolated and its nucleotide

sequence can be determined. Controlled sequences of DNA can be identified and

analyzed. Protein/Enzyme/RNA function can be investigated. Mutations can be identified, e.g. gene defects related

to specific diseases. Organisms can be engineered foe specific purposes,

e.g., Insulin production.

FUTURE PROSPECTS OF MOLECULAR CLONING

• In the near future, molecular cloning will likely see the emergence of a new paradigm, with synthetic biology techniques that will enable in vitro chemical synthesis of any in silico-specified DNA construct.

• These advances should enable faster construction and iteration of DNA clones, accelerating the development of gene therapy vectors, recombinant protein production processes and new vaccines.

REFERENCES

1. Hall, RM; Collis, CM (1995). "Mobile gene cassettes and integrons: Capture and spread of genes by site-specific recombination". Molecular microbiology 

2. Plant biotechnology – the genetic manipulations of plants 2nd edition by Adrian Slater, Nigel W.Scott & Mark R. Fowler3. Principles of gene Manipulations & Genomics – Primrose &

Twyman 7th edition4. Joseph Sambrook, David Russell. "Chapter 1". Molecular

Cloning - A Laboratory Manual 1 (3rd ed.)5.  Lederberg, J and Lederberg, EM (1952) Replica plating and

indirect selection of bacterial mutants. J Bacteriol. 6. Wikipedia search