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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