lmjs培训-链霉菌遗传操作和pcr-targeting-sep2011.ppt

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2017-06-28 发布于广东
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lmjs培训-链霉菌遗传操作和pcr-targeting-sep2011.ppt

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lmjs培训-链霉菌遗传操作和pcr-targeting-sep2011

* * * * * * * * * * * ... into a recombinant molecule 3′ 5′ 5′ 3′ PCR-targeting (step 1) S. coelicolor cosmid neo SuperCos1 bla target X X 39bp 39bp P1 P2 marker oriT FRT FRT PCR product S. coelicolor cosmid neo bla P1 P2 marker oriT FRT FRT PCR-targeting (step 2) target S. coelicolor chromosome S. coelicolor cosmid neo SuperCos1 bla X X P1 P2 marker oriT FRT FRT P1 P2 marker oriT FRT FRT S. coelicolor chromosome Why two-step strategy? ? Red is efficient in E. coli Recombinant cosmid-DNA can easily be confirmed by PCR, restriction analysis and/or sequencing Mutagenised cosmids can be mobilised by conjugation, no need for transformation procedures High frequency of double cross-overs due to long flanking sequences in a cosmid clone Disadvantages Dependent upon the availability of a E. coli clone Not high throughput (in comparison to transposon mutagenesis) DC SC Conjugation plate Template cassettes for gene replacements P1 P2 aac(3)IV oriT FRT FRT P1 P2 aac(3)IV oriT loxP loxP P1 P2 oriT aac(3)IV SwaI SwaI pIJ773 pIJ774 pIJ775 P1 P2 aadA oriT FRT FRT P1 P2 neo oriT P1 P2 aadA FRT FRT P1 P2 neo FRT FRT pIJ776 pIJ777 pIJ778 pIJ779 FRT FRT P1 P2 vph oriT FRT FRT P1 P2 vph FRT FRT pIJ780 pIJ781 vph P1 P2 tet oriT FRT FRT pIJ782 P1 P2 hyg oriT FRT FRT pIJ797 NEW Template cassettes for other applications attP bla bla tet int oriT pIJ787 P1 tipAp P2 oriT FRT FRT aac(3)IV pIJ785 P1 tcp P2 oriT FRT FRT aac(3)IV pMS80 P1 P2 aac(3)IV oriT FRT FRT nitAp fd-ter NEW Herai et al., 2004. Hyper-inducible expression system for streptomycetes. PNAS 101, 14031-14035 P1 P2 aac(3)IV oriT FRT FRT egfp pIJ786 bla bla aac(3)IV oriT pIJ784 bla bla hyg oriT pIJ798 NEW neo neo aac(3)IV aac(3)IV pIJ789 pIJ794 oriT neo neo neo neo neo neo oriT oriT aadA vph pIJ795 pIJ796 NEW S. coelicolor cosmid neo bla REDIRECT (Rapid Efficient Directed Recombination Time saving)Download protocol and Primer design program at .uk/redirect/index.html To obtain the REDIRECT