expression vector Replicon and Promoter Region
Components of expression vector
- Ribosome Binding Site
- Selection Marker
- Multiple Cloning Site
1. Low Copy Number
(for 15-20 copy per cell – e.g. pBR22)
2. High Copy Number
(for 500-700 copy per cell) (e.g. pUC18)
Typically, high-copy plasmids are employed for the expression of proteins and cloning process. In some cases, a high-copy may cause a problem in the process of DNA cloning. For example, cloned DNA may encode proteins that show toxicity when expressed in high levels. Even if a protein is cloned at low levels, the presence of large copies of the plasmid gene may increase the level of the protein to toxic levels. In these cases, the use of lower-numbered plasmids can reduce the level of protein to below the toxicity concentrations. It should also be borne in mind that due to the metabolic limitation, having a high copy number decreases the growth of the bacterium and may also lead to instability of the plasmid, and therefore the number of intact organisms expressing the protein would be diminished.
For this reason, the use of high-copy plasmids does not result in increased protein production. In the case of requirement to use two plasmids simultaneously, they should be compatible with their initiator region. In other words, the plasmids belonging to the same group should not transfect the host at the same time. In the below Table, some of the most commonly used cloning plasmids and the type of their Ori-region are listed that include a promoter region called Ori and Cis-acting regulatory elements. Genetic elements that can be spontaneously integrated, such as the plasmid, have Replicon. One of the critical parameters to consider when selecting a suitable vector is the number of copies. The control of the copy number of the plasmid depends on its Ori region.
Plasmids are classified into the following groups based on their Ori area :
The promoter causes the expression of the protein and includes the promoter (the position of RNA polymerase) and the operator. The promoter gene sequence controls the binding of RNA polymerase and transcription factors. The promoter in the plasmid should be compatible with the type of RNA supposed to be synthesized. For example, The RNA Polymerase II promoter is used to synthesize mRNA, and the RNA Polymerase III Promoter is used to synthesize shRNA. Also, the promoter you choose should be compatible with the host cell, because the transcription system varies in cells and organisms, and the promoter uses the host transcription system.
Some of the most common bacterial promoters used in cloning :