pET vector. Plasmid for Expression by T7 RNA Polymerase.

Plasmid Expression Vector System. It cantian the T7 promoter.

1. pET Vector Muhammad Mujahid ID: F2019253023

2. vector In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA There are two types of Vector. Cloning vector. Expression vector.

3. How Expression vector differ from Cloning vector? The first question to ask yourself is what you intend to do with your vector. Cloning vectors are useful for generating many copies of your gene. Expression vectors are associated with the actual expression of the gene into mRNA and protein in the target organism. Cloning vectors usually contain features associated with the insertion or removal of DNA fragments. For example, they have multiple cloning sites with many restriction sites, antibiotic resistant genes, etc. Expression vectors, however, contain additional organism specific sequences relating to expression such as promoters, RBS sequences, Kozak sequences (in eukaryotes), or the Shine Dalgarno sequence (in prokaryotes).

4. Why the pET system is powerful system? Vectors with strong, controllable promoters are used to maximize the synthesis of cloned gene product But recombinant DNA technology is mainly used for synthesis of large quantities of protein, either to study its properties or because it has commercial value . To maximize gene expression only selecting the strongest promoter possible is not enough, as the effects of over expression on the host cell also need to be considered. Over expression of a protein may be toxic to the host cell . High-level synthesis can also exert metabolic drain on the cell leading to slower growth. To minimize the problems associated with high-level expression, it is usual to use a vector in which the cloned gene is under the control of a regulated promoter.

5. Introduction of pET vector pET - Plasmid for Expression by T7 RNApolymerase . Originally constructed by Studier and colleagues. Size 5700 bp. These are a family of expression vectors that utilize phage T7 promoters to regulate synthesis of cloned gene products. Derived from the pBR322 plasmid, pET vectors engineered to take advantage of the features of the T7 bacteriophage gene 10 that promote high-level transcription and translation. It is one of the most widely used systems for the cloning and in vivo expression of recombinant proteins in E . coli .

6. Components of pET vector.

7. Components of pET Vector. T7 promoter: Drives high-level transcription of the gene of interest when T7 RNA polymerase is present. When placed immediately upstream of a LacO element, the entire cassette is known as the T7lac promoter. LacO: Binding site for LacI. This element inhibits activity of the T7 promoter when LacI protein is present, preventing leaky expression of the gene of interest. RBS: The ribosome-binding site and translation initiation element from T7 bacteriophage. This allows for efficient production of the protein of interest. ORF: The open reading frame of your gene of interest is placed here. T7 terminator: Signal sequence to terminate the transcript made from the gene of interest, preventing run-on transcription. Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli. pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin as well as the Rop gene exist in low copy numbers in E. coli. Rop: Repressor of primer. It encodes a small protein that regulates plasmid copy number . The presence of the Rop protein, in combination of pBR322 origin of replication on the plasmid, results in low copy numbers of the plasmid. LacI: The E. coli natural promoter and coding sequence for the lac repressor. In the absence of induction of the system (i.e. without IPTG), the LacI protein represses transcription of the gene of interest from the T7lac promoter, as well as transcription of T7 RNA polymerase from the LacUV5 promoter in host strains used for recombinant protein production.

8. Regulation of expression of Gene Cloned into a pET vector The gene for T7 RNApolymerase (gene 1) is inserted into the chromosome of E. coli and transcribed from the lac promoter; therefore, it will be expressed only if the inducer IPTG is added. Normal function - no protein expression (LacI protein represses transcription by blocking T7 RNA polymerase expression). Altered function- protein expression (IPTG binds to Lac repressor protein and expresses T7 RNA polymerase for transcription). The T7 RNA polymerase will then recognize the T7 promoter on the vector and transcribe the gene cloned into the pET vector. If the protein product of the cloned gene is toxic, it may be necessary to further reduce the transcription of the cloned gene before induction. The T7 lysozyme encoded by a compatible plasmid, pLysS, will bind to any residual T7 RNApolymerase made in the absence of induction and inactivate it. Also, the presence of lac operators between the T7 promoter and the cloned gene will further reduce transcription of the cloned gene in the absence of the inducer IPTG.

9. Advantages The T7 promoter is one of the strongest known promoters. It can produce a lot of protein. The pET plasmids have many common restriction sites, especially in front of the T7 promoter but also in other places. The pET plasmids have a medium copy number. (~20-25 per cell), which can be helpful because it prevents weird things from happening due to copy numbers that are too high or too low. It allows for the high expression level of the T7 promoter without overloading the cell with many copies of the plasmid in addition. Strong expression:  The T7 transcription and translation regulatory system allows for very high-level production of proteins of interest. Tightly controlled expression:  The expression of the gene of interest is generally very strongly repressed in the absence of added IPTG. So, the pET expression system is easily controlled by Inducer (IPTG)

10. Disadvantages Host requirements:  Completed pET vectors should be maintained in an E. coli strain lacking the T7 RNA polymerase gene, and must be transferred to a separate host strain containing the T7 RNA polymerase gene before induction of protein expression. Potential leaky expression in some hosts:  Even in the absence of IPTG, there may be some low-level expression of T7 RNA polymerase from the Lac promoter in some expression host strains, which could lead to bacterial toxicity for certain genes of interest in certain host strains.

11. Uses of pET vector

12. References S.B. Primrose and R.M. Twyman. “Principles of Gene Manipulation and Genomics”. Seventh Edition Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. “Use of T7 RNA polymerase to direct expression of cloned genes”. Methods Enzymol. 1990;185:60-89 Studier FW, Moffatt BA. “Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes”. J Mol Biol. 1986 May 5;189(1):113-30. Dubendorff JW, Studier FW. "Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor". Journal of Molecular Biology. 1991 ;219 (1): 45–59.