expression vectors and promoters

1. Promoters usedin ExpressionVectors
Presented By
Nasir ul islam
Class : 6th semester
Roll no : 180847
Reg no. :Cus-18-spc-10074
Subject : Lab Techniques in Applied Biotechnology
TEACHER INCHARGE
DR SADIQ MAJEED

2. Expression vectors
Introduction
 A vector used for expression of cloned DNA fragment in a
host cell is called as an expression vector.
 These vectors are frequently engineered to contain
regulatory sequences that act as promoter and/or enhancer
regions and lead to efficient transcription of the insert gene.
 Two elements that are required for active gene expression: a
strong promoter and a ribosome binding site near an initiating
ATG codon.
 Expression vectors are used for molecular biology techniques
such as site directed mutagenesis.
 The main function of an expression vector is to yield the
product of gene, therefore a strong promoter is necessary.
The more mRNA is produced, the more protein is made.

3. Types of Expression Vectors
PROKARYOTIC EXPRESSION VECTOR EUKARYOTIC EXPRESSION VECTOR
Bacterial expression system (e.g. E. Coli) Yeast expression system (e.g. S.
Viral expression system (e.g. Baculovirus)
Mammalian cell expression system

4. Expression in E.coli
 E.coli is one of the most widely used expression host for protein expression.
 The techniques for expression in E.coli are well developed and work by increasing
the number of copies of the gene or increasing the binding strength of the
promoter region so assisting transcription.
 For example a DNA sequence for a protein of interest could be cloned into a high
copy no plasmid containing the lac promoter, which is then transformed into the
bacterium Escherichia coli. Addition of IPTG (a lactose analog) activates the lac
promoter and causes the bacteria to express the protein of interest.
 Advantages:
• Rapid doubling time (approximately 30 minutes)
• Growth in simple defined media and inexpensive
• Proteins of both prokaryotes and eukaryotes origin can be produced within the organism.

5. In expression vector various Genetic elements required are:
1. Origin of replication
2. Selective markers
3. Transcriptional promoters
4. Unique multiple cloning sites
5. Transcriptional initiation regions (TIRs)
6. Translational terminator

6.  Origin of replication:
 An origin of replication is a sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. For
small DNAs, including bacterial plasmids and small viruses, a single origin is sufficient.
 The origin of replication determines the vector copy number, which could typically be in the range of 25–50
copies/cell if the expression vector is derived from the low-copy-number plasmid pBR322, or between 150 and 200
copies/cell if derived from the high-copy-number plasmid pUC. The copy number influences the plasmid stability, i.e.
the maintenance of the plasmid within the cells during cell division. A positive effect of a high copy number is the
greater stability of the plasmid when the random partitioning occurs at cell division. On the other hand, a high number
of plasmids generally decreases the growth rate, thus possibly allowing for cells with few plasmids to dominate the
culture, since they grow faster.
 Selective Marker
 The selectable marker is the sequence on DNA which helps in identifying and elimination of non transformants and
selectively permitting the growth of transformants.
 For the maintenance of the Plasmid in the cell, a selectable marker is necessary.
 Selectable markers are often antibiotic resistance genes .
 Normally the genes encoding resistance to antibiotics such as Ampicillin, Chloroamphenicol, tetracycline or kanamycin,
etc., are considered useful selectable markers for E. coli

7.  Transcriptional promoter:
promoter is a –
• Region of DNA that control the transcription of a particular gene.
• It locate upstream of the genes .
• Recognizing site for the RNA polymerase (sigma subunit)
Good promoter should be :
o Strong promoter: accumulation of expressed protein up 10 to 30 % or more of the total cellular
protein.
o Minimal basal expression level: Tight regulation of the promoter.
o Easily induction: simple and cost effective manner.
Multiple cloning site :
 It is defined as a short segment of DNA which contain many restriction site (usually 20+ )
 To simplify the insertion of the heterologous gene in the correct orientation within the vector.

8. Translation initiation regions (TIRs)
 Located on the 5- mRNA
Translational terminator :
 located on the 3- mRNA
 Use of Rho-independent termination
 Many different promoter sequences have been used to illicit inducible protein production
in E.coli
1. lac promoter
2. tac promoter
3. λpl promoter
4. T7 Expression system

9. The control of gene expression
 Each cell in human contains all the genetic material for the growth and development of
a human
 Some of these genes will be need to be expressed all the time .
 Other genes are not expressed all the time
 They are switched on an off at need
The lac operon
 An operon is a group of genes that are transcribed at the same time.
 They usually control an important biochemical process and found in prokaryotes
 The lac operon consists of three genes each involved in processing the sugar lactose
 One of the is the gene for the enzyme β-galactosidase (hydrolysis lactose into glucose
and galactose )

10. Cont…

11. The control of the lac operon
The lac promoter:
 Lac promoter provides a mechanism for inducible gene expression.
 Without lactose in the cell ,the repressor proteins continuously synthesised. It sits on a sequence
of DNA just in front of the lac operon, the operator sites
 The repressor protein blocks the promoter site where the RNA polymerase settles before it starts
transcribing
 With lactose in the cell, a small amount of a sugar allolactose is formed within the bacterial
cell. This fits onto the repressor protein at another active site (allosteric site)
 This causes the repressor protein to change its shape (a conformational change ). It can no
longer sit on the operator site . RNA polymerase can now reach its promoter site .
 When glucose and lactose are present RNA polymerase can site on the promoter site but its unstable and keeps it falling

12. The tac promoter
 The tac promoter is weak because the 35 region deviates from the consensus .
 The creation of a fusion sequence containing the -35 region of the E.coli trp
operon and the -10 region of the lac operon controlling the expression of the
genes responsible for tryptophan biosynthesis and lactose metabolism which
results in the formation of the tac promoter
 The tac promoter is 5 times stronger the lac promoter.
 Expression vectors carry the tac promoter also carry the lacO operator and
usually the lacl gene coding the lac repressor .
 Because of this , these vectors are ITPG inducible and can be repressed and
induced in a variety of E. coli strains.

13. Cont…
 DNA sequence of lac , trp , and tac promoter
 The consensus E.coli -35 and -10 sequence based on the analysis of naturally occurring
promoters are shown and the sequence of each of the promoter, extending from the -
35 region to translational start site , are shown .
 The tac promoter is the hybrid of the trp and lac promoter .
 The -35 and -10 region contains closely resemble the consensus sequence .
 The tac promoter is able to induce the expression of target genes such that encoded
polypeptide can accumulate at the level of 20-30%of the totalcell protein
Thank you