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.
i. Cloning vector.
ii. 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.
i. Normal function- no protein expression (LacI protein represses
transcription by blocking T7 RNA polymerase expression).
ii. 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.

10. 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)

11. 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.

12. Uses of pET vector

14. References
1. S.B. Primrose and R.M. Twyman. “Principles of Gene
Manipulation and Genomics”. Seventh Edition
2. 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
3. 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.
4. 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.