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Cloning and rDNA (II) Dr. Abdulaziz Almalik National center for Biotechnology King Abdulaziz City for Science and Technology Office: 228-Building 17 (F) Tel: 4813154 [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 vector 2. Understand the function of each element Expression vector Palindrome DNA of interest Restriction Enzyme Delivery vector Expressio system Expressio system Recombinant DNA Expression vector An 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 Plasmid • 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 pUC18 Selectable marker a gene (antibiotic resistance) when expressed on plasmid will allow host cells to survive Multiple Cloning Site (MSC) also called Polylinker is a short segment of DNA which contains many (up to ~20) restriction sites Promoter is a short DNA sequence which enhances expression of adjacent gene Ori origin of replication is a particular sequence where replication is initiated 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 pUC18 Selectable marker a gene (antibiotic resistance) when expressed on plasmid will allow host cells to survive Multiple Cloning Site (MSC) also called Polylinker is a short segment of DNA which contains many (up to ~20) restriction sites Promoter is a short DNA sequence which enhances expression of adjacent gene Ori origin of replication is a particular sequence where replication is initiated 2 4 1 1. 2. 3. 4. 5. 6. 5 Isolation of gene of interest Integration into expression vector Transformation into host cells Growth of cells (fermentation) Isolation & purification of protein Formulation of protein product 6 You are now able to: Recognize the main elements of an expression vector Understand the function of each element