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Expression vector, pBAD vector

This expression vector lecture explains the use of pBAD vector in gene cloning. http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-m... An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.[1] The goal of a well-designed expression vector is the production of significant amount of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for biotechnology and the production of proteins, for example insulin which is important for medical treatments of diabetes. The expression host of choice for the expression of many proteins is Escherichia coli as the production of heterologous protein in E. coli is relatively simple and convenient, as well as being rapid and cheap. A large number of E. coli expression plasmids are also available suitable for a wide variety of needs. Other bacteria used for protein expression include Bacillus subtilis. Most proteins are expressed in the cytoplasm of E. coli, but where necessarily, for example when the protein can only fold correctly in an oxidizing environment due to the presence of disulphide bonds, the protein may be targeted to the periplasmic space by the use of an N-terminal signal sequence. Other more sophisticated systems are being developed; such systems may allow for the expression of proteins previously thought impossible in E. coli, such as glycosylated proteins.[2][3] The promoters used for these vector are usually based on the promoter of the lac operon or the T7 promoter,[4] and they are normally regulated by the lac operator. These promoters may also be hybrids of different promoters, for example, the tac promoter is a hybrid of trp and lac promoters.[5] Note that most commonly used lac or lac-derived promoters are based on the lacUV5 mutant which is insensitive to catabolite repression. This mutant allows for expression of protein under the control of the lac promoter when the growth medium contains glucose since glucose would inhibit protein expression if wild-type lac promoter is used.[6] Presence of glucose nevertheless may still be used to reduce background expression through residual inhibition in some systems.[7] Examples of E. coli expression vectors are the pGEX series of vectors where glutathione-S-transferase is used as a fusion partner and protein expression is under the control of the tac promoter,[8][9][10] and the pET series of vectors which uses a T7 promoter.[11] It is possible to simultaneously express two or more different proteins in E. coli using different plasmids. However, when 2 or more plasmids are used, each plasmid needs to use a different antibiotic selection as well as a different origin of replication, otherwise the plasmids may not be stably maintained. Many commonly-used plasmids are based on the ColE1 replicon and are therefore incompatible with each other; in order for a ColE1-based plasmid to coexist with another in the same cell, the other would need to be of a different replicon, e.g. a p15A replicon-based plasmid such as the pACYC series of plasmids.[12] Another approach would be to use a single two-cistron vector or design the coding sequences in tandem as a bi- or poly-cistronic construct.[13][14] Yeast A yeast commonly used for protein expression is Pichia pastoris.[15] Examples of yeast expression vector in Pichia are the pPIC series of vectors, and these vectors use the AOX1 promoter which is inducible with methanol.[16] The plasmids may contain elements for insertion of foreign DNA into the yeast genome and signal sequence for the secretion of expressed protein. Proteins with disulphide bonds and glycosylation can be efficiently produced in yeast. Another yeast used for protein expression is Kluyveromyces lactis and the protein is expressed driven by a variant of the strong lactase LAC4 promoter.[17] Saccharomyces cerevisiae is also commonly used for protein expression, for example in yeast two-hybrid system for the study of protein-protein interaction.[18] The vectors used in yeast two-hybrid system contain fusion partners for two cloned genes that allow the transcription of a reporter gene when there is interaction between the two proteins expressed from the cloned genes. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia. Link- http://en.wikipedia.org/wiki/Main_Page