Plasmids and vectors.pdf

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7/31/13 Biology 305 Laboratory: Plasmids and vectors

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Plasmids and vectors

A B O U T P L A S M I D S A N D 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-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 Databasecurrently has information for more than 4000

vectors.

Common features of vectors used for cloning DNA fragments include

small sizehigh 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 vitrotranscription

P L A S M I D/ V E C T O R M A P S

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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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 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

P G E M - T E A S Y

For a map of this vector, please see the Promegasite.

I S O L A T I N G P L A S M I D D N A

S t r a t e g y

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 o l u m e o f b a c t e r i a l c e l l s n e e d e 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 or

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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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.

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Plasmids and vectors.pdf