Genetic Engineering Lecture 3 Vectors PDF

Vector    Vectors Vectors  A vector is a small piece of DNA molecule, which is used as a vehicle to carry foreign DNA into host cell, where it can be replicated and/or expressed.  The most common vectors that used in rDNA technology taken from a virus o...

Vector    Vectors Vectors  A vector is a small piece of DNA molecule, which is used as a vehicle to carry foreign DNA into host cell, where it can be replicated and/or expressed.  The most common vectors that used in rDNA technology taken from a virus or a plasmid of bacteria Recombinant DNA:  A vector containing foreign DNA is termed as recombinant DNA. It has DNA from different organism.  Insertion of a vector into the target cell is usually called transformation for bacterial cells, and transfection for eukaryotic cells. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 1  Vectors Figure: rDNA technology using different types of vector Essential Characteristics of a Vector  Capable of self-replication inside the host cell.  Has restriction sites (cloning region) for cutting by restriction enzyme and inserting target DNA.  Is a small size in comparison to the host chromosome for ease of isolation.  It is circular (or more accurately a closed loop), so that it is less likely to be broken down  It contain marker genes, so that cells containing the vector can be identified.  They should be easily isolated from host cell. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 2  Vectors Genetic Markers  These are needed to identify cells that have successfully taken up a vector and so become transformed.  above less than 1% of the cells actually take up the vector, so a marker is needed to distinguish these cells from all the others.  A common marker, used in plasmids, is a gene for resistance to an antibiotic such as tetracycline.  Bacterial cells taking up this plasmid are resistant to this antibiotic.  So if the cells are grown on a medium containing tetracycline all the normal untransformed cells (99%) will die.  Only the 1% transformed cells will survive, and these can then be grown and cloned on another plate. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 3  Vectors Types of vectors Vectors can be classified into: cloning vectors and expression vectors Cloning vector  The purpose of the cloning vector is to carry foreign DNA to the host.  The cloning vector does not necessarily help to express a protein which the foreign DNA encodes.  Depending on the size and the application of the insert the suitable vector is selected for a particular purpose. Types of Cloning Vectors 1. Plasmids  A common choice of vector.  Plasmids are small, circular DNA molecules that are found in the cytoplasm of bacteria and separate from the rest of the chromosome.  They replicate independently of the bacterial chromosome.  Useful for cloning DNA, inserts less than 20 kb (kilobase pairs).  Inserts larger than 20 kb are lost easily in the bacterial cell.  Often synthetic plasmids are used that contain: o an origin of replication, where replication starts/ initiates Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 4  Vectors o a selectable marker (often for antibiotic resistance) o cloning site(s) which can be cut by restriction enzymes for the insertion of foreign DNA.  They are found widely in many bacteria, for example in E. coli, but may also be found in a few eukaryotes, for example in yeast such as Saccharomyces cerevisiae.  Plasmid regions with multiple adjacent cloning sites (i.e. a cluster of restriction sites) are called polylinkers.  Plasmids are inserted into bacterial cells by a process called transformation. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 5  Vectors 2. Bacteriophage lambda (λ)  Bacteriophage are viruses that infect bacteria.  They can multiply inside bacterial cell by using some or all of the bacteria enzymes.  Bacteriophages have a very high significant mechanism for delivering its genome into bacterial cell. Hence it can be used as a cloning vector to deliver larger DNA segments.  Most of the bacteriophage genome is non-essential and can be replaced with foreign DNA.  Using bacteriophage as a vector, a DNA fragment of size up to 20 kb can be transformed Bacteriophage lambda (λ)  The linear double-stranded DNA lambda genome contains about 50,000 nucleotide pairs and encodes 50-60 different proteins.  When the lambda DNA enters the cell the ends join to form a circular DNA molecule.  The bacteriophage can multiply in E. coli by a lytic pathway, which destroys the cell, or it can enter a latent prophage state.  Damage to a cell carrying a lambda prophage induces the prophage to exit from the host chromosome and shift to lytic growth (green arrows). Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 6  Vectors  The entrance and exit of the lambda DNA from the bacterial chromosome are site-specific recombination events. 3. Cosmids  Cosmids are hybrids of phages and plasmids that can carry DNA fragments up to 45 kb.  They can replicate like plasmids but can be packaged like phage lambda. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 7  Vectors 4. Bacterial artificial chromosomes (BACs)  Bacterial artificial chromosomes (BACs) are simple plasmid.  Designed to clone very large DNA fragments ranging in size from 75 to 300 kb.  BACs basically have marker like sights such as antibiotic resistance genes and a very stable origin of replication (ori). 5. Yeast artificial chromosomes (YACs)  Can be grown in E.coli and Yeast  Miniature chromosome  Can accept 200 kb -3000 kb; useful for sequencing  Mostly YACs are used for cloning very large DNA fragments and for the physical mapping of complex genomes.  YACs have an advantage over BACs in expressing proteins that require post translational modifications. 6. Human artificial chromosomes (HACs)  Human artificial chromosomes (HACs) or mammalian artificial chromosomes (MACs) are still under development.  HACs are micro-chromosomes that can act as a new chromosome in a population of human cells. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 8  Vectors  HACs range in size from 6 to 10 Mb that carry new genes introduced by human researchers.  HACs can be used as vectors in transfer of new genes, studying their expression. Table: Comparison of vectors generally available for cloning DNA fragments Vector Host cell Vector structure Insert range (k b) M13 E. coli Circular virus 1-4 Plasmid E. coli Circular plasmid 1-5 Phage 2, E. coli Linear virus 2-25 Cosmids E. coli Circular plasmid 35-45 BACs E. coli Circular plasmid 50-300 YACs S. cerevisiue Linear 100-2000 chromosome Expression vector  A vector designed specifically for the transcription and protein expression of the transgene in the target cell is called expression vector, and generally have a promoter sequence that drives expression of the transgene. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 9  Vectors Other types of vectors Shuttle Vectors  Shuttle vector is a cloning vector capable of replicating in two or more types of organism (e.g., E. coli and yeast).  Shuttle vectors may replicate autonomously in both hosts, or may integrate into the host genome. Ti vectors  Ti vectors are widely used to transfer genes into plants  Ti vectors are naturally occurring plasmids (around 200 kb in size)  Isolated from the bacterium Agrobaderiumtumefaciens, Which is a soil borne plant pathogen that causes a condition in plants called crown gall disease  When A. tumefaciensenters host plants, a piece of DNA (T-DNA) from the Ti plasmid (Ti stands for tumor- inducing) inserts into the host chromosome  T-DNA encodes for the synthesis of a hormone called auxin, which weakens the host cell wall Infected plant cells divide and enlarge to form a tumor (gall)  Plant geneticists recognized that if they could remove auxin and other detrimental genes from the Ti plasmid, the resulting vector could be used to deliver genes into plant cells. Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 10  Vectors Figure: Transfected plant using T DNA vector Genetic Engineering BIOC0402 Lecture 3 Dr. Fatema S Alatawi 11