Promoters used in expression vectors

expression vectors and promoters

1. Promoters used in Expression Vectors Presented By Nasir ul islam Class : 6 th 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. cervesiae) 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: Origin of replication Selective markers Transcriptional promoters Unique multiple cloning sites Transcriptional initiation regions (TIRs) 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 : Strong promoter: accumulation of expressed protein up 10 to 30 % or more of the total cellular protein. Minimal basal expression level: Tight regulation of the promoter. 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 lac promoter tac promoter λ pl promoter 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