1、Designation:E1493 06Standard Guide forIdentification of Bacteriophage M13 or Its DNA1This standard is issued under the fixed designation E1493;the number immediately following the designation indicates the year oforiginal adoption or,in the case of revision,the year of last revision.A number in pare
2、ntheses indicates the year of last reapproval.Asuperscript epsilon()indicates an editorial change since the last revision or reapproval.INTRODUCTIONThis guide covers the identification of bacteriophage M13 or its DNA and was developed by TaskGroup E48.02.03 on viruses.The objective is to describe la
3、boratory characterization procedures thatwould be sufficient to verify that a biological preparation believed to contain M13(or M13 DNA)foruse in any step of a biotechnology process actually does contain this bacteriophage(or its DNA).This guide assumes a basic knowledge of microbiology and molecula
4、r biology.1.Scope1.1 This guide covers the identification of bacteriophageM13 used in biotechnology.1.2 There are many variants of M13 that have been devel-oped specifically for cloning technology.These variants haveforeign DNA inserted into the M13 genome,causing the M13to differ in size and genoty
5、pe.1.3 If the M13 is to be used to construct a recombinantmolecule,then the criteria described in Section 6 should beused to characterize the newly made DNA.2.Terminology2.1 Definitions:2.1.1 alpha complementationthe ability of a short amino-terminal fragment(alpha fragment)of b-galactosidase to for
6、ma functional complex with the carboxyl terminal fragment(omega fragment).2.1.2 F1an F factor that contains a portion of the E.coligenome.2.1.3 F+factoran episome of E.coli.Encoded on it arethe functions necessary to produce an F pilus.2.1.4 F pilusa protrusion on E.coli that is necessary formating.
7、The F pilus also contains the receptor for phage M13.2.1.5 Hfra strain of E.coli in which the F+factor isinserted into the chromosome.2.1.6 multiple cloning siteDNA that contains severalcontiguous restriction enzyme recognition sites;also called apolylinker.2.1.7 phage displaythe use of bacteriophag
8、e M13 or avariant to insert a peptide sequence fused to a coat protein ofM13.3.Significance and Use3.1 This guide is intended for use in a biotechnologylaboratory when the need arises to identify a preparationcontaining M13 bacteriophage or DNA.4.Background Information about M13 Bacteriophage4.1 M13
9、 is a filamentous bacteriophage that infects male(F+,F1or Hfr)Escherichia coli.The phage particles containcircular single-stranded DNA,6407 nucleotides in length,coated with the protein product of the M13 gene 8.(1)24.2 The phage attaches to a receptor at the end of the F pilus.The infecting single-
10、stranded DNA(+strand)replicates in thecell:a complementary()strand is synthesized,resulting in adouble-stranded,replicative form(RF).Using the RF as atemplate,both strands can be replicated,increasing the copynumber of the RF to about 20 to 40 per cell.Late in infection(+)strands are preferentially
11、produced and packaged intophage particles for export from the cell.(1)4.3 M13 infection and subsequent phage release is not lethalto Escherichia coli.Bacteriophage DNA is continually repli-cated and packaged,causing a decrease in the growth rate ofthe host.The“plaque”seen upon infection by M13 is th
12、e resultof an area of decreased growth rate,not actually cell killing.(1)4.4 M13 has extensive sequence homology to bacterio-phages f1 and fd,differing in only a few bases.(1)4.5 M13 is used in biotechnology often as a vector intowhich foreign DNA can be cloned.Commonly used M131This guide is under
13、the jurisdiction of ASTM Committee E55 on Manufactureof Pharmaceutical Products and is the direct responsibility of Subcommittee E55.04on General Biopharmaceutical Standards.Current edition approved Nov.1,2006.Published November 2006.Originallyapproved in 1992.Last previous edition approved in 2001
14、as E1498 01.DOI:10.1520/E1493-06.2The boldface numbers in parentheses refer to a list of references at the end ofthis standard.1Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959,United States.Copyright ASTM International Provided by IHS under license with
15、 ASTM Licensee=Ohio State University/5967164005,User=ahmadi,rozitaNot for Resale,03/26/2012 04:36:50 MDTNo reproduction or networking permitted without license from IHS-,-,-variants are the M13 mp series.These M13 bacteriophageshave a portion of the E.coli lac operon with a multiple cloningsite with
16、in a truncated lacZ gene.4.5.1 A portion of the E.coli lac operon containing the3-prime end of the lacI gene,the lac promoter and operator,and the alpha complementation portion of the lacZ gene isinserted near the origin of replication of the M13.(2,3,4)4.6 M13 or variants are used in phage display for theselection of proteins or peptides with improved or novelbinding characteristics.Typically,a DNAlibrary containing thepotential coding sequences for the peptides is cloned into oneof the genes f
