Journal:

Article Title: Membrane Topology of the Streptomyces lividans Type I Signal Peptidases

doi: 10.1128/JB.183.16.4752-4760.2001

Figure Lengend Snippet: Plasmids used in this work

Article Snippet: Oligonucleotides used in this study table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Plasmid Relevant properties Reference or source E. coli pGEM-T Easy Used for cloning of PCR amplified DNA fragments, Ap r Promega pDR540 Contains the galK gene under control of the tac promoter, Ap r Accession no. {"type":"entrez-nucleotide","attrs":{"text":"U13847","term_id":"595699","term_text":"U13847"}} U13847 pSN40 pRM1 derivative (30) containing an S. lividans TK21 chromosomal Bam HI fragment encoding the four SPases, Ap r 32 pEX50 pDR540 derivative, tac promoter, Ap r This work pET3a T7 promoter, Ap r Novagen pEX20 pET3a derivative encoding hexahistidine-tagged SipY336 This work pEX23 pET3a derivative encoding hexahistidine-tagged SipY304 This work pTM pGEM-T Easy derivative containing the gene encoding mature tendamistat This work pSipTM pTM derivative, contains different SPase-TM fusion genes This work pEXSipY336 pEX50 derivative encoding wild-type SipY This work pEXSipY304 pEX50 derivative encoding SipY derivative lacking the C-terminal anchor This work Streptomyces pAX5a E. coli-Streptomyces shuttle vector encoding pre-tendamistat, Ap r Ts r 15 pIJ6021 Contains a multiple cloning site after a tipA promoter, Km r 39 pIJSipTM pIJ6021 derivative encoding the different SPase-TM fusion proteins This work Open in a separate window Plasmids used in this work Colony hybridization S. lividans colonies were grown on nylon filters (Hybond N+; Amersham Pharmacia Biotech) placed on agar plates containing kanamycin (50 μg/ml) at 27°C for 2 days.

Techniques: Clone Assay, Amplification, Plasmid Preparation

Journal:

Article Title: Membrane Topology of the Streptomyces lividans Type I Signal Peptidases

doi: 10.1128/JB.183.16.4752-4760.2001

Figure Lengend Snippet: Schematic representation of pEX50 and pTM. (A) pEX50 was constructed with the primers DR-1 and DR-2 by using plasmid pDR540 DNA. After cleavage with NcoI, the amplified fragment containing the introduced restriction sites for NdeI, NcoI, and BamHI was self-ligated, resulting in the removal of the galK gene. (B) pTM was constructed by inserting the gene encoding the mature part of tendamistat, which was amplified by PCR with the primers MaTM5′ and TM3′ by using pAX5a as a template, into the pGEM-T Easy plasmid. The resulting vector was used for the construction of SPase-tendamistat gene fusions (SipTM).

Article Snippet: Oligonucleotides used in this study table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Plasmid Relevant properties Reference or source E. coli pGEM-T Easy Used for cloning of PCR amplified DNA fragments, Ap r Promega pDR540 Contains the galK gene under control of the tac promoter, Ap r Accession no. {"type":"entrez-nucleotide","attrs":{"text":"U13847","term_id":"595699","term_text":"U13847"}} U13847 pSN40 pRM1 derivative (30) containing an S. lividans TK21 chromosomal Bam HI fragment encoding the four SPases, Ap r 32 pEX50 pDR540 derivative, tac promoter, Ap r This work pET3a T7 promoter, Ap r Novagen pEX20 pET3a derivative encoding hexahistidine-tagged SipY336 This work pEX23 pET3a derivative encoding hexahistidine-tagged SipY304 This work pTM pGEM-T Easy derivative containing the gene encoding mature tendamistat This work pSipTM pTM derivative, contains different SPase-TM fusion genes This work pEXSipY336 pEX50 derivative encoding wild-type SipY This work pEXSipY304 pEX50 derivative encoding SipY derivative lacking the C-terminal anchor This work Streptomyces pAX5a E. coli-Streptomyces shuttle vector encoding pre-tendamistat, Ap r Ts r 15 pIJ6021 Contains a multiple cloning site after a tipA promoter, Km r 39 pIJSipTM pIJ6021 derivative encoding the different SPase-TM fusion proteins This work Open in a separate window Plasmids used in this work Colony hybridization S. lividans colonies were grown on nylon filters (Hybond N+; Amersham Pharmacia Biotech) placed on agar plates containing kanamycin (50 μg/ml) at 27°C for 2 days.

Techniques: Construct, Plasmid Preparation, Amplification