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1、PCR targeting system in Streptomyces coelicolor A3(2)Bertolt Gust, Tobias Kieser and Keith Chater, John Innes Centre, Norwich Research Park, Colney, Norwich NR47UH, UK, Tel: +44 (0)1603 452751 Fax: +44 (0)1603 456844IntroductionMany bacteria are not readily transformable with linear DNA because of t
2、he presence of the intracellular recBCD exonuclease that degrades linear DNA. However, the 入 RED ( gam, bet, exo) functions promote a greatly enhan ced rate of recomb in ati on when using linear DNA. By exploiting this, Datsenko and Wanner (2000) made 40 different disruptions on the E. coli chromoso
3、me by replacing the wild-type sequences with a selectable marker generated by PCR using primers with 36 nt homology extensions.The strategy for PCR-targeting for mutagenesis of Streptomyces coelicolor is to replace a chromosomal sequence within a S. coelicolor cosmid (Redenbach et al., 1996) by a se
4、lectable marker that has been generated by PCR using primers with 39 nt homology extensions. The inclusion of oriT (RK2) in the disruption cassette allows conjugation to be used to introduce the PCR targeted cosmid DNA into S. coelicolor. Conjugation is much more efficient than transformation of pro
5、toplasts and it is readily applicable to many actinomycetes (Matsushima et al., 1994). The potent methylspecific restriction system of S. coelicolor is circumvented by passaging DNA through a methylation-deficient E. coli host such as ET12567 (MacNeilet al., 1992). Vectors containing oriT (RK2; Pans
6、egrau et al., 1994) are mobilisable in trans in E. coli by the self-transmissible pUB307 (Bennett et al., 1977, Flett et al., 1997) or the non-transmissible pUZ8002, which lacks acis-acting function for its own transfer (Kieser et al., 2000).To adapt the procedure of X RED mediated recomb in ati on
7、for Streptomyces cassettes for gene disruptions were constructed that can be selected both in E. coli and in Streptomyces(Table 1). After a single disruption with an oriT-containing cassette, further disruptions can be performed on the same cosmid using oriT-free cassettes containing alternative sel
8、ective markers. The X RED recombination plasmid pKD20(E. coli Genetic Stock Center CGSC Strain # 7637) was modified by replacing the ampicilli n resista nee genebla with the chloramphe ni col resista nee gene cat,gen erat ing plJ790, to permit selecti on in the prese nee of Supercosl-derived cosmids
9、 (ampicilli n and kan amyci n resista nee).Name of plasmidResista nce- markerResista neeConcen tratio n for E. colioriTSize of templatepIJ773Fig. 5aac(3)IV apramycin50 旧/ml LB+ 1332bppIJ778Fig. 6aadAspect ino myci n streptomyci n50 旧/ml LB50(jg/m LB+ 1425bppIJ779.aadAspect ino myci n- streptomyci n5
10、0 旧/ml LB50 旧/ml LB-1057bppIJ780Fig.7vph viomycin30 jjg/ml DNA+ 1497bppIJ781vph viomycin30 jjg/ml DNA-16：!2bpTable 1 : Disrupti on cassettes containing differe nt resista nee markers with and without oriT: All disruption cassettes were cloned into theEccRV site of pBluescript SK II (+) allowing the
11、isolation of a EcoRI/Hi ndlll fragme nt for use as template for the PCR reacti on. The size of the cassettes in eludes the 19 bp and 20 bp primer site (see section 2:“ primer design ” ) which are identical in all disruptioncassettes. The resista nee genes with or withoutriT are flan ked by FRT sites
12、 (FLP recog niti on targets)which allows FLP-mediated excisi on of the cassette (see sect ion 7:“ FLP-mediated excisi on of thedisruption cassette ” ).#Fig. 1: Flowchart of gene disruption by PCR-targetingAmplification of the disruption cassetteTemplates for PCR amplification1384 bp1424 bpPaac1496 b
13、pin duction of 入 RED by L-arab inosetran sformati onPCR-targetingloss of temperature sen sitive 入 RED recomb in ati on plasmid pIJ790 over ni ght at 37 CLegend:aac(3)IV : apramycin resistance gene aadAAti nomyci n/streptomycin resista nce gene bet, exo : promote recomb in ati oncat : chloramphenicol
14、resistance geneFLP: FLP-recombinaseConjugati on withFRT : FLP recognition targetgam : inhibits the host RecBCD exonuclease V neo : kanamycin resistance geneORF: open reading frameS.coelicolororiT : origin of transfer from RK2 ts : temperature-sensitive replicon vph : viomycin resistance geneTran sfo
15、rmati on ofE.colicontaining FLP systemstrainE.coli ET12567 / pUZ8002 / cosmidConjugative transfer and screening for double cross-oversFLP-mediated excision ofdisruption cassettein duce FLP syn thesis and loss of temperature sen sitive FLP recomb in ati on plasmid BT340 over ni ght亠gene disrupti ons can be repeated using the same markerStreptomycesdisrupti on muta ntStreptomyces in frame deleti onn OALa rnr0 snatProtocol (see Flowchart Fig. 1)Puri
