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Vectors (Bio Tech)

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21 views

Vectors (Bio Tech)

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Green
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© © All Rights Reserved
Available Formats
Download as PDF or read online on Scribd
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2.4 Vectors Vector is an autonomously replicating genetic element used to carry a fragment of target DNA into a host ceil for the purpose of cloning and expression. The term vector refers to the DNA molecule that act as transporting Vehicle which carry foreign DNA from the test tube to the host cell. Cloning vectors are used to clone foreign DNA whereas expression vectors are engineered so that any foreign DNA can be transcribed in RNA and translated into protein. A viral DNA or plasmid is generally used as a cloning vector. The important features of a cloning vector are as follows: 1. Ability to replicate in host cells. 2. Genetic marker to select for host cells containing the vector. 3. Unique restriction enzyme sites for insertional cloning. 4. Minimum amount of non-essential DNA. Vectors for E.coli 1. Cloning vectors based on plasmid Plasmids are naturally occurring circular, extrachromosomal, autonomously replicating DNA, present in many prokaryotic and few eukaryotic organisms. Plasmids range in size from approximately 1Kbp to over 300 kbp. aa c- w~ wns weston oo Oey IEF relaxed OF Stringent OM TNE Dass Wt Wie ties we come we Plasmid that are maintained within the cel. Relaxed plasmids are maintlned at multiple copies per cel, white Stringent plasmids are present at a snale copy, oF alow number of coples per cel. Piasmids can be categorized into one of two major types ~ conjugative or non-conjugative - depending upon whether or not they carry a set of transfer genes, called the tra genes, whieh promate bacterial conjugation, Plasmids encode only a few of the proteins required for thelr own replication. Most of the proteins required for replication are provided by the host cel. Those repiation proteins that are plasmid-encoded are located Very close to the origin of replication sequences. Origin of replication also determines the host range of a Plasmid. Thus, only a small region surrounding the origin of replication site Is required for replication. Other Parts of the plasmid can be deleted and foreign sequences can be added to the plasmid and replication will Stil cceur Plasmid vectors provide & simple way of coring small ONA fragments in bacterial (and simple eukaryotic) ‘ells, The Ideal plasmid cloning vector must have small size, high copy number, own origin of replication, ‘estrction sites for many restriction enzymes and selectable markers, Ths, in order to adapt natural plasmid ‘molecules a8 cloning vectors, several modiiations are normally made ‘+ nsertion of a multiple cloning ske painter. This isa short (~30 bp) synthetic sequence which contains unique restriction stes fora variety of common restcion nodeases (pre-existing restriction stes for these enzymes wil be deleted from the plasmid f necessary to ensure the presence of unique doning ses) Insertion ofan antbiotic resitance gene. The host cels that are used must naturally be sensitive to the Antibiot In question so that eny vector molecule which transforms a host cell ean confer antibiotic resistance. By plating transformed ces on a medium containing the antibiot, only those cells that have been transformed by vector roles survive ‘+ Insertion of a selection system for screening of recombinants, Typcaly this involves arranging for the ‘multiple cloning site polylinker to be inserted into an expressible gene or gene fragment within the plasmid ‘An example of a widely used closing vector 's pBR322, which replicates in Ecol. In pBR322, p stands for plasmid and BR stands for ts developer Solver and Rodriguez, "322" distinguishes this plasmid trom others

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