ASTM E1493-01
(Guide)Standard Guide for Identification of Bacteriophage M13 or Its DNA
Standard Guide for Identification of Bacteriophage M13 or Its DNA
SCOPE
1.1 This guide covers the identification of bacteriophage M13 used in biotechnology.
1.2 There are many variants of M13 that have been developed specifically for cloning technology. These variants have foreign DNA inserted into the M13 genome, causing the M13 to differ in size and genotype.
1.3 If the M13 is to be used to construct a recombinant molecule, then the criteria described in Section 6 should be used to characterize the newly made DNA.
General Information
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Designation: E 1493 – 01
Standard Guide for
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Identification of Bacteriophage M13 or Its DNA
This standard is issued under the fixed designation E1493; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
This guide covers the identification of bacteriophage M13 or its DNAand was developed by Task
Group E48.02.03 on viruses. The objective is to describe laboratory characterization procedures that
would be sufficient to verify that a biological preparation believed to contain M13 (or M13 DNA) for
use in any step of a biotechnology process actually does contain this bacteriophage (or its DNA).
This guide assumes a basic knowledge of microbiology and molecular biology.
1. Scope 3. Significance and Use
1.1 This guide covers the identification of bacteriophage 3.1 This guide is intended for use in a biotechnology
M13 used in biotechnology. laboratory when the need arises to identify a preparation
1.2 There are many variants of M13 that have been devel- containing M13 bacteriophage or DNA.
oped specifically for cloning technology. These variants have
4. Background Information about M13 Bacteriophage
foreign DNAinserted into the M13 genome, causing the M13
4.1 M13 is a filamentous bacteriophage that infects male
to differ in size and genotype.
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1.3 If the M13 is to be used to construct a recombinant (F,F or Hfr) Escherichia coli. The phage particles contain
circular single-stranded DNA, 6407 nucleotides in length,
molecule, then the criteria described in Section 6 should be
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used to characterize the newly made DNA. coated with the protein product of the M13 gene 8.
4.2 ThephageattachestoareceptorattheendoftheFpilus.
2. Terminology
The infecting single-stranded DNA(+strand) replicates in the
2.1 Definitions: cell: a complementary (−) strand is synthesized, resulting in a
2.1.1 alpha complementation—the ability of a short amino- double-stranded, replicative form (RF). Using the RF as a
terminal fragment (alpha fragment) of b-galactosidase to form template, both strands can be replicated, increasing the copy
a functional complex with the carboxyl terminal fragment number of the RF to about 20 to 40 per cell. Late in infection
(omega fragment). (+) strands are preferentially produced and packaged into
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2.1.2 F—an F factor that contains a portion of the E. coli phage particles for export from the cell.
genome. 4.3 M13 infection is not lethal to Escherichia coli. Bacte-
2.1.3 F factor—anepisomeof E. coli.Encodedonitarethe riophage DNAis continually replicated and packaged, causing
functions necessary to produce an F pilus. a decrease in the growth rate of the host. The “plaque” seen
2.1.4 F pilus—a protrusion on E. coli that is necessary for upon infection by M13 is the result of an area of decreased
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mating. The F pilus also contains the receptor for phage M13. growth rate, not actually cell killing.
2.1.5 multiple cloning site—DNA that contains several 4.4 M13 has extensive sequence homology to bacterioph-
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contiguous restriction enzyme recognition sites; also called a ages f1 and fd, differing in only a few bases.
polylinker. 4.5 M13 is used in biotechnology often as a vector into
2.1.6 phage display—the use of bacteriophage M13 or a which foreign DNA can be cloned. Commonly used M13
variant to insert a peptide sequence fused to a coat protein of variants are the M13 mp series. These M13 bacteriophages
M13. haveaportionofthe E. colilacoperonwithamultiplecloning
site within a truncated lacZ gene.
4.5.1 A portion of the E. coli lac operon containing the
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3-prime end of the lacI gene, the lac promoter and operator,
ThisguideisunderthejurisdictionofASTMCommitteeE48onBiotechnology
and is the direct responsibility of Subcommittee E48.02 on Characterization and
Identification of Biological Systems.
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Current edition approved May 10, 2001. Published July 2001.
Rasched, I., and Oberer, E., Ff coliphages, Structural and Functional Relation-
Originally published as E 1498–92. Last previous edition E 1498–92 (1998).
ships, Microbiological Reviews, Vol 50, 1986, pp. 401–427.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
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E 1493
and the alpha complementation portion of the lacZ gene is are grown with vigorous aeration for 5 to 8 h. Longer times
3,4,5
inserted near the origin of replication of the M13. will result in fewer phage particles. The bacterial cells are
4.6 M13 or variants are used in phage display for the centrifuged out and the bacteriophage remain in the superna-
selection of p
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