A type of cloning vector that has a sequence on the 5’ side of the multiple cloning site that initiates transcription is called an expression vector (Miesfeld 1999). Expression vectors have DNA sequences that surround the multiple cloning site and serve as specific binding sites for transcription factors (Miesfeld 1999). For the study of gene expression in cultured cells and transgenic organisms, there are four main types of promoters used in expression vectors (Miesfeld 1999). These four main types are minimal promoters, strong constitutive promoters, cell-specific promoters, and regulated promoters (Miesfeld 1999).
The minimal promoters contain a multiple cloning site in front of the TATA box. These usually serve as reporter genes for insertion of transcriptional regulator sequences (Miesfeld 1999). The strong constitutive promoters can be used in nearly all cell types (Miesfeld 1999). As the name suggests, these promoters are used for high level (strong) gene expression (Miesfeld 1999). Cell-specific promoters are used to initiate expression in specific cell types (Miesfeld 1999). Regulated promoters are controlled by transcription factors that are activated via some sort of stimulus, such as hormones (Miesfeld 1999). Minimal promoters can be used in almost all eukaryotic cells; however, the other three promoters are more cell-specific and often used in studies with transgenic organisms (Miesfeld 1999).
Summary of Lecture Notes
Expression vectors require both transcription and translation which sets it apart from transcription only vectors. First, the plasmid, as well as the DNA to be inserted, is cut with a restriction enzyme
. The cut DNA and plasmids are mixed together in the presence of ligase to repair the nicks between the DNA and the plasmid. When the coding region of a gene is placed at the site of insertion in the proper orientation and secured through ligation, the inserted gene will be transcribed into mRNA as well as translated into protein by the host cell (Watson et al 2008). The promoter in the expression vector can be chosen so that expression of the insert is regulated by the addition of a simple compound to the growth medium (Watson et al 2008). E.coli
is commonly used as a host.
Applications and Scientific Basis¶
Numerous expression vectors differ by their promoter sequence, which can be deliberately controlled to accomplish the desired expression. This characteristic allows for significant amounts of mRNA to be assembled which can be translated into the desired proteins to be analyzed (Sambrook & Russell 2001).
Two typical types of promoters include strong promoters and tightly regulated promoters (Clark 2005). A strong promoter for expression vectors paired with a particular gene can be used to synthesize a specific desired protein (Watson et al 2008). This is the most common use of expression vectors. When wanting to control the expression of the cloned gene during production of a large quantity of proteins, it is especially effective to use a strong promoter (Clark 2005).
When using a tightly regulated promoter, a gene and its expression can be tested for uses in biotechnology and genetic engineering (Clark 2005). This way of testing a gene is often done in growth media with controlled conditions and variables. The promptness and regulation of the expression will be affected by changing the components that are added to the growth medium. The ability to control the growth medium affects when a particular gene will be expressed (Watson et al 2008).
Along with the promoter sequence, vectors may also contain ribosome binding sites, which control the expression of the cloned gene (Clark 2005).
Expression vectors can also be used to express and research mutant genes to determine what their function is and what can be caused or fixed by the mutation (Watson et al 2008).
Expression vectors also have been specifically used in gene therapy for type II diabetes (Parson et al 2007).
Clark, D.P. (2005). Molecular biology: Understanding the genetic revolution. Burlington, MA: Elsevier Academic. (pp. 631-33).
Miesfeld, R.L. (1999). Applied molecular genetics. New York, NY: Wiley-Liss, Inc. (pp. 176-77).
Parsons, G.B. & Souza, D.W. & Wu, H. & Yu, D. et al. (2007). Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes. Gene Therapy. Genzyme Corporation: Framingham, MA. Vol. 14, Iss. 1, pp. 38-48.
Sambrook, J. & Russell, D.W. (2001). Molecular cloning: A laboratory manual. 3rd ed. Vol. 1. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. (pp. 1.13-1.14).
Watson, J. D. & Baker, T. A. & Bell, S. P. et al. (2008). Molecular biology of the gene. 6th ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. (pp 567 & 747).
Plasmid: Expression Vector
This is a diagram of a plasmid containing an expression vector. The diagramed expression vector uses the lacI promoter that is classified as being a strong promoter. In such instances, an induced molecule, IPTG, is added to promote transcription which then removes itself from the DNA allowing RNA polymerase to bind. If IPTG is absent then the lacI repressor does not allow the gene to be expressed (Clark 2005).