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Bi ol og y 305 Labor ator y: Plasmids and vector s
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Plasmids and vectors AB O U T PLASMIDS
AND V E C T O R S
The terms plasmid and vector are often used interchangeably, but their meanings are slightly different. A plasmid is an extra-chr extra-chromosomal omosomal DNA D NA molecule found found in bacteria (Sambrook and Russell, Ru ssell, 2001 [1]). Plasmids and chromosomes are replicated rep licated using the the same sa me enzym enzymes, es, but plasmids are replicated replic ated and a nd inherited inherited independent independently ly from from the bacterial chromosomes. Normally Normally a bacterium bacteri um will will have have only one one copy co py of its chromosome but it can have multiple multiple copies cop ies of a plasmid. In nature, plasmids usually carry gene(s) that are beneficial to, but not absolutely required by, the cell in which which they reside reside.. For example, bacterial antibiotic antibio tic resistance resi stance genes are often carried on plasmids. For more detailed background on plasmids and vectors please see [ 1]. A vector, vector, in molecu molecular lar biology biology,, refers refers to a plasmid th that is engineer engineered ed to make it a more more usefu usefull tool tool for molecular molecular biologis bi ologists ts (all vectors vectors are plasmids plasmids,, but not all plasmids are vectors). Vectors are designed desi gned for a variety of applications applic ations incl i ncluding uding easy cloning cloning of foreign DNA and easy expressi expression on of foreign proteins. The The database databas e Vector Database currently Database currently has information for more than 4000 vectors. Common features of vectors used for cloning DNA D NA fragments include include small size high copy number number (number (number of plasmids maintained per cell) marker gen gene(s) for easy selection selectio n in bacteria or other hosts hosts a multiple multiple cloning site/region site/regi on (MCS (MCS or MCR) – a short section sectio n of DNA containing several unique restriction enzyme recognition sequences visual marker marker for selecting selecti ng bacteria carryin carrying g plasmids plasmi ds with wi th inserts inserts (blue white white screening) s creening) promoters for in vitro transcription vitro transcription
P L A S M I D / V E C T O R
MAPS
For cloning purposes, we need to know what what features features a vector has has and their relative positions posi tions in the th e vector. We represent rep resent this this information visually visually using a plasmid plasmi d or vector map, which which is is a cartoon representation, drawn to scale, sca le, showing showing the relative positions posi tions of key k ey cloning cloning features. Generally Gen erally such such maps are constr co nstructed ucted using the plasmid's DNA sequence. In maps, plasmid bases b ases are numbered numbered sequen seq uentially tially,, in a clockwise clock wise fashion, fas hion, starting starting with base 1 and ending on th the e base immedi immediately ately coun counter-clockwise ter-clockwise to base ba se 1. That is, if a plasmid plasmi d is i s 3000 base pair p air (bp) in i n size it will have have bases numbered numbered 1 – 3000. Map posi p ositions tions of various vector vector features featur es are indicated i ndicated relative to their distance dis tance from base 1. For example, in the the map of pUC18 pUC18,, the cut site for EcoR I is at a t position posi tion 396, and for Sap SapII is at position 690. bio305l ab.wiki dot.com/pr i nter - - fr iendl y//r esour ces:aboutplasmi ds
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Biology 305 Laboratory: Plasmids and vectors
U s i n g p la s m i d m a p s One use for plasmid maps is to predict the sizes of restriction enzyme products. For example, cutting pUC18 with EcoR I and SapI at the same time will generate two linear DNA pieces. One piece would contain bases 396 to 690 and would be 294 bp in length. The second piece would contain the rest of the plasmid, from position 690 all the way around to position 396 for a total length of 2392 bp. Plasmid map practice problems
PGEM®-T
E AS Y
For a map of this vector, please see the Promega site.
ISOLATING
PLASMID
DNA
Strategy Bacteria actually do most of the work in plasmid DNA production. Bacteria are usually grown to stationary phase in liquid media so as to produce the maximum amount of plasmids per ml of culture. As the bacteria grow and divide they also replicate the plasmids that we force them to carry. Plasmids are then isolated from the cultures. There are many protocols for isolating plasmid DNA from bacterial cells but they all contain the same two basic steps: lyse the cells and separate the plasmid DNA from the other cell components. Usually cells are lysed either using heat, or using alkaline conditions. Once the cells are lysed, the plasmid DNA is separated from the rest of the cellular components using a combination of chemical and physical techniques. This sounds a lot more complicated than it is.
Volume of bacterial cells needed The amount of culture needed depends on the approximate yield of plasmid DNA per milliliter of culture. Plasmid yield per ml is primarily dependent on the plasmid copy number (the average number of plasmids per cell). Plasmid copy number is controlled by a plasmid's replicon, which includes the DNA replication origin (the ori) and DNA encoded replication control elements. In plasmids, the ori and regulatory elements are usually found close together. More than 30 different plasmid replicons have been described but almost all plasmids used in molecular cloning carry a replicon derived from pMB1 (Sambrook and Russell, 2001). The naturally occurring pMB1 replicon has a copy number of 15 to 20 plasmids per bacterial cell. However, vectors derived from pMB1, such as the pUC family or the pGEM family, carry highly bio305lab.wikidot.com/printer--friendly//resources:aboutplasmids
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Biology 305 Laboratory: Plasmids and vectors
modified replicons that can be maintained at much higher copy numbers. For example, the plasmid pKC7 is a pBR322 derivative that carries the un-altered pMB1 replicon and under normal conditions, is maintained at 15 to 20 copies per cell. On the other hand, pGEM-T ®, is maintained at 500 to 700 copies per cell.
Citations 1. Sambrook, J. and D. Russell. 2001. Plasmids and their usefulness in molecular cloning. In: Molecular Cloning: A Laboratory Manual , Vol. 1, 3rd ed. CSH Press, Cold Spring Harbor, NY. p. 1.2-1.29 view source print page revision: 21, last edited: 29 Oct 2012, 06:08 (275 days ago)
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