These vectors are needed in biotechnology for the synthesis of recombinant protein from genes that are not expressed correctly when cloned in E. They have special origin of replication called as autonomously replicating sequences (ARS), e.g., yeast replicative plasmid vectors (YRp) etc. plasmid vectors, bacteriophages vectors, cosmids, phasmids, phagemids, etc. Bacteria support different kinds of vectors, e.g. These are special bacterial origin of replication and antibiotic resistance selectable markers. For example, if we are making a vector for a bacterial host, it must have a suitable origin of replication which will be functional in a bacterial cell.ĭepending on this basis the vectors are classified as under: All the parts of the vectors must be functionally compatible with the host. So depending on the host cell the vectors are designed and constructed. (c) The bacterial polypeptide may also help in purification of the target polypeptide by different purification techniques such as affinity chromatography.Īfter construction of a recombinant DNA these can be introduced into a host cell. products of ompA genes), then our target polypeptide will simply be transported outside of the host cell straight into the culture media from where these can be collected. For example, if the bacterial peptides are derived from a protein that is exported by the cell (e.g. (b) The bacterial polypeptide may act as a signal peptide, responsible for transporting our target protein to a specific location from where these are collected. In contrast the foreign polypeptides that lack a bacterial segment are often destroyed. (a) The presence of bacterial peptide at the start of fusion protein may stabilize the molecule and prevent it from being degraded by the host cell. The followings are the reasons for incorporation of a fusion protein before our gene of interest: This hybrid polypeptide chain consisting of two different types of polypeptides is called a fusion protein. The product of gene expression is therefore a hybrid protein, consisting of short bacterial polypeptide fused into amino terminus of our target polypeptide sequence. In this way we fuse two reading frames, producing a hybrid gene that starts with the bacterial gene and progresses without a break into the codons of our gene of interest. While using such type of expression vectors the gene of interest is inserted just after the gene for bacterial polypeptide. coli), multiple cloning site is not immediately adjacent to the ribosome binding sequence, but instead is preceded by a special sequence coding for a bacterial polypeptide. In some types of expression vectors which are specifically used in association with the bacterial host (like E. Sequences that control transcription initiation, such as regulator genes and operators. Prokaryotic transcription initiation and termination sequences.ĭ. A DNA sequence that, when transcribed into RNA, produces a prokaryotic ribosome binding site.Ĭ. The promoter precedes a restriction site where foreign DNA is to be inserted, allowing transcription of foreign sequence to be regulated by adding substances that induce the promoter.ī. A bacterial promoter, such as the lac promoter. To get the protein we need to allow the expression of our gene of interest (hence the name expression vector) by employing the processes of transcription and translation.Īpart from the three DNA sequences discussed above (origin of replication, selectable markers and multiple cloning sites), the expression vectors have some special additional sequences as well.Ī. We use an expression vector when our aim is to obtain the protein product of our gene of interest. Expression Vectors or Expression construct: Example: PUC cloning vectors, pBR322 cloning vectors, etc.Ģ. In all cases, the vector needs to be genetically modified in order to accommodate the foreign DNA by creating an insertion site where the new DNA will fitted. Some are created synthetically, as in the case of yeast artificial chromosomes and bacterial artificial chromosomes, while others are taken from bacteria and bacteriophages. A number of organisms can be used as sources for cloning vectors. These are mostly used in construction of gene libraries. We use a cloning vector when our aim is to just obtain numerous copies (clones) of our gene of interest (hence the name cloning vectors). The point is what we are targeting from our gene of interest - its multiple copies or its protein product.ĭepending on these criteria vectors are of following two types: On the Basis of Our Aim with Gene of Interest: On the Basis of Cellular Nature of Host Cell. On the Basis of Our Aim with Gene of Interest 2.
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