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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.
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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.
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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. coli colonies transformed with plasmids carrying the desired foreign cloned DNA segment.The classifications are: 1. In this manner, the presence of lac Z gene in pUC and resistance genes against ampicillin and tetracycline in pBR322 allow selection of E. The plasmids carrying no insert on the other hand, will be able to grow in media containing one or both the antibiotics. The insert bearing plasmid can be selected by their ability to grow in a medium containing only one of the two antibiotics and by their failure to grow in a medium containing both the antibiotics. As discussed above, in pBR322, the DNA is inserted at a site located in one of the two genes for resistance against antibiotics, so that it will inactivate one of the two resistance genes. These cloning vectors are available in pairs with reversed orders of restriction sites relative to lac Z promoter pUC 8 and pUC 9 make one such pair (Fig.
#Difference between plasmid and cosmid series
Another series of plasmids that are used as cloning vectors belong to pUC series (after the place of their initial preparation i.e. coli plasmid ColEl), which is 4,362 bp DNA and was derived by several alterations in earlier cloning vectors (Fig. One of the standard cloning vectors widely used in gene cloning experiments is pBR322 (derived from E. In such a situation, the parent vector in bacterial cells can be selected by resistance against two antibiotics and the chimeric DNA can be selected by retention of resistance against only one of the two antibiotics.
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If a plasmid has two such genes conferring resistance against two antibiotics and if the foreign DNA insertion site lies within one of these two genes, then the chimeric vector loses resistance against one antibiotic, the gene for which has foreign DNA inserted within its structure. Selection of chimeric DNA is also facilitated by the resistance genes which the plasmid may carry against a certain antibiotic. A foreign DNA segment can now be inserted, by joining the ends of broken circular DNA to the two ends of foreign DNA, thus regenerating a bigger circular DNA molecule that can now be separated by gel electrophoresis on the basis of its size (Fig.
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