cloning and rdna (ii) dr. abdulaziz almalik national center for biotechnology king abdulaziz city...
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Cloning and rDNA (II)
Dr. Abdulaziz AlmalikNational center for Biotechnology
King Abdulaziz City for Science and TechnologyOffice: 228-Building 17 (F)
Tel: [email protected]
Objectives of this lecture
By the end of this lecture you will be able to:1. Recognize the main elements of an
expression vector2. Understand the function of each element
Expressionvector
Expressionvector
DNA of interestDNA of interest
Restriction Enzyme
Restriction Enzyme
Recombinant DNA
Recombinant DNADelivery vectorDelivery vector
PalindromePalindrome
Expressio system
Expressio system
Expressio system
Expressio system
Expression vectorAn expression vector, is usually a plasmid designed to carry the gene of interest to be inserted
in an expression system (i.e. host cells).
An expression vector (i.e. plasmid) is engineered to contain regulatory sequences regions (e.g. promoter), an origin of replication, a selectable marker, and a suitable site for the insertion of a
gene of interest such as the multiple cloning site.
efficient transcription of the gene carried on the expression vector.
the production of significant amount of messenger RNA, and therefore proteins.
Plasmid
• Extrachromosomal DNA molecules
• Usually transfer antibiotic-resistance between bacteria
• Why plasmid?
• Can be engineered to carry a variety of inserts.• The ability to obtain many copies of plasmid (i.e. cloning) through normal
division of the host cells. • Provide a cheap and easy way of mass-production of proteins in an
expression system.
• Expression system: cells that receive the rDNA and can use it to produce the product of interest.
• Insertion of a vector into the target cell is usually called transformation for bacterial cells, transfection for eukaryotic cells, although insertion of a viral vector is often called transduction
Plasmid
Multiple Cloning Site (MSC) also called Polylinker is a short segment of DNA which contains many (up to ~20) restriction sites
Ori origin of replication is a particular sequence where replication is initiated
Promoter is a short DNA sequence which enhances expression of adjacent gene
Selectable marker a gene (antibiotic resistance) when expressed on plasmid will allow host cells to survive
pUC18
Multiple Cloning Site
• A short segment of DNA which contains many (up to ~20) restriction site for several restriction enzymes.
• These restriction sites are different sequences that when they are cut by restriction enzymes, they represent DNA sequences that a complementary foreign DNA fragments can be inserted into
Promoter
• A region in the DNA that initiates the transcription of a particular gene
Promoter
• Activator protein binds to enhancer sequence and attracts proteins to the promoter region that activate RNA polymerase leading to transcription
Lac Operon
• Bacteria organize multiple genes under the control of one promoter. The set of genes plus promoter is called an operon
• The lac operon of E. coli is a segment of DNA that includes a promoter, an operator, and the three structural genes that code for lactose-metabolizing enzymes
Origin of Replication
• A sequence of DNA at which replication is initiated.
• A portion of the double helix is unwound by a helicase.
• A molecule of a DNA polymerase binds to one strand of the DNA and assembling a complementary strand of nucleotides and reforming a double helix.
Selectable Marker
• A gene introduced to a cell that provides a property for artificial selection e.g. antibiotic resistance
Multiple Cloning Site (MSC) also called Polylinker is a short segment of DNA which contains many (up to ~20) restriction sites
Ori origin of replication is a particular sequence where replication is initiated
Promoter is a short DNA sequence which enhances expression of adjacent gene
Selectable marker a gene (antibiotic resistance) when expressed on plasmid will allow host cells to survive
pUC18
11221. Isolation of gene of interest2. Integration into expression vector3. Transformation into host cells4. Growth of cells (fermentation)5. Isolation & purification of protein6. Formulation of protein product
1. Isolation of gene of interest2. Integration into expression vector3. Transformation into host cells4. Growth of cells (fermentation)5. Isolation & purification of protein6. Formulation of protein product
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You are now able to: Recognize the main elements of an
expression vector Understand the function of each element