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Biology 305 Laboratory: Plasmids and vectors

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Plasmids and vectors

ABO UT PL AS M IDS
AND VECT O RS
The terms plasmid and vector are often used interchangeably, but their meanings are slightly different. A plasmid is an extra-chromosomal DNA molecule found in bacteria (Sambrook and Russell, 2001 [1]). Plasmids and chromosomes are replicated using the same enzymes, but plasmids are replicated and inherited independently from the bacterial chromosomes. Normally a bacterium will have only one copy of its chromosome but it can have multiple copies of a plasmid. In nature, plasmids usually carry gene(s) that are beneficial to, but not absolutely required by, the cell in which they reside. For example, bacterial antibiotic resistance genes are often carried on plasmids. For more detailed background on plasmids and vectors please see [1]. A vector, in molecular biology, refers to a plasmid that is engineered to make it a more useful tool for molecular biologists (all vectors are plasmids, but not all plasmids are vectors). Vectors are designed for a variety of applications including easy cloning of foreign DNA and easy expression of foreign proteins. The database Vector Database currently has information for more than 4000 vectors. Common features of vectors used for cloning DNA fragments include small size high copy number (number of plasmids maintained per cell) marker gene(s) for easy selection in bacteria or other hosts a multiple cloning site/region (MCS or MCR) a short section of DNA containing several unique restriction enzyme recognition sequences visual marker for selecting bacteria carrying plasmids with inserts (blue white screening) promoters for in vitro transcription

PL ASM ID/ VECT O R

M APS

For cloning purposes, we need to know what features a vector has and their relative positions in the vector. We represent this information visually using a plasmid or vector map, which is a cartoon representation, drawn to scale, showing the relative positions of key cloning features. Generally such maps are constructed using the plasmid's DNA sequence. In maps, plasmid bases are numbered sequentially, in a clockwise fashion, starting with base 1 and ending on the base immediately counter-clockwise to base 1. That is, if a plasmid is 3000 base pair (bp) in size it will have bases numbered 1 3000. Map positions of various vector features are indicated relative to their distance from base 1. For example, in the map of pUC18, the cut site for EcoRI is at position 396, and for SapI is at position 690.
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Biology 305 Laboratory: Plasmids and vectors

U s ing pla s mid ma ps

One use for plasmid maps is to predict the sizes of restriction enzyme products. For example, cutting pUC18 with EcoRI 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 ASY
PL ASM ID

For a map of this vector, please see the Promega site.

I SO L AT ING
Strategy

DNA

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.

V olume of ba c te ria l c e lls ne e de d

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
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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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