puc19 cloning vector; lacz ap r  (Promega)

Journal:

Article Title: Expression of the cpdA Gene, Encoding a 3?,5?-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex ▿ †

doi: 10.1128/JB.01350-08

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: All medium components were purchased from Difco, and chemicals and antibiotics were obtained from Sigma. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains V. vulnificus ATCC 29307 Clinical isolate; virulent 14 AR ATCC 29307 but spontaneous rifampin resistance 26 HY101 AR but Δ cpdA 20 KP301 AR but Δ cya 20 KC74 ATCC 29307 but crp :: nptI 14 E. coli DH5α (φ80 lacZ ΔM15) recA1 endA1 gyrA96 relA1 thi-1 hsdR17 (r K − m K − ) supE44 deoR Δ( lacZYA-argF ) U169 Laboratory collection SM10λpir thi-1 thr leu tonA lacY supE recA ::Rp4-2-Tc::Muλ pir ; OriT of RP4; Km r 30 JM109 endA1 recA1 gyrA96 thi-1 hsdR17 (r K − m K − ) relA1 supE44 Δ( lac-proAB ) [F′ traD3 6proAB lacI q Z ΔM15] Promega Plasmids pUC19 Cloning vector; lacZ Ap r 34 pINE45 pUC19 with 1.72-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pINE45-1 pUC19 with 4.3-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pLAFR5 IncP Tc r ; derivative of pLAFR3 containing double cos cassettes 16 pHS51 pLAFR5 containing EcoRI and HindIII fragment of pINE45 This study pQE30 Expression vector; Ap r Qiagen pQE30-cpdA pQE30 with V. vulnificus cpdA This study pHK0011 Transcriptional fusion plasmid with promoterless luxAB ; Tc r 8 pSMK-cpdA-1 pHK0011 with cpdA upstream region (positions −1521 to +41 relative to cpdA IC) This study pSMK-cpdA2 pHK0011 with cpdA upstream region (positions −1178 to −1051 relative to cpdA IC) This study pSMK-cpdA3 pHK0011 with cpdA upstream region (positions −1163 to −1051 relative to cpdA IC) This study pGEM-11zf(+) General cloning vector; Ap r Promega pGEM-11zf(+)-cpdA2mt pGEM-11zf(+) with the mutagenized sequence for the putative CRP-binding site of cpdA regulatory region This study pSMK-cpdA2mt pSMK-cpdA2 but mutagenized CRP-binding site This study Open in a separate window Strains and plasmids used in this study

Techniques: Plasmid Preparation, Clone Assay, Expressing, Sequencing

Journal:

Article Title: Expression of the cpdA Gene, Encoding a 3?,5?-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex ▿ †

doi: 10.1128/JB.01350-08

Figure Lengend Snippet: Determination of the transcriptional start site of the cpdA gene. (A) Primer extension using V. vulnificus RNA and oligonucleotide primer yqiE-R (annealing to positions +130 to +150 relative to the IC of mutT). Lanes C, T, A, and G represent sequencing ladders of pINE45-1. The +1 indicates the site of transcriptional initiation. (B) Sequence of the upstream region of mutT, the first gene of the operon composed of mutT, yqiB, cpdA, and yqiA. The initiation codon for mutT is in boldface type, and the promoter and the putative −10 and −35 regions are underlined. The putative binding site for the cAMP-CRP complex is designated with a box. The primers used for the construction of the cpdA::luxAB transcription fusions are indicated with horizontal arrows.

Article Snippet: All medium components were purchased from Difco, and chemicals and antibiotics were obtained from Sigma. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains V. vulnificus ATCC 29307 Clinical isolate; virulent 14 AR ATCC 29307 but spontaneous rifampin resistance 26 HY101 AR but Δ cpdA 20 KP301 AR but Δ cya 20 KC74 ATCC 29307 but crp :: nptI 14 E. coli DH5α (φ80 lacZ ΔM15) recA1 endA1 gyrA96 relA1 thi-1 hsdR17 (r K − m K − ) supE44 deoR Δ( lacZYA-argF ) U169 Laboratory collection SM10λpir thi-1 thr leu tonA lacY supE recA ::Rp4-2-Tc::Muλ pir ; OriT of RP4; Km r 30 JM109 endA1 recA1 gyrA96 thi-1 hsdR17 (r K − m K − ) relA1 supE44 Δ( lac-proAB ) [F′ traD3 6proAB lacI q Z ΔM15] Promega Plasmids pUC19 Cloning vector; lacZ Ap r 34 pINE45 pUC19 with 1.72-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pINE45-1 pUC19 with 4.3-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pLAFR5 IncP Tc r ; derivative of pLAFR3 containing double cos cassettes 16 pHS51 pLAFR5 containing EcoRI and HindIII fragment of pINE45 This study pQE30 Expression vector; Ap r Qiagen pQE30-cpdA pQE30 with V. vulnificus cpdA This study pHK0011 Transcriptional fusion plasmid with promoterless luxAB ; Tc r 8 pSMK-cpdA-1 pHK0011 with cpdA upstream region (positions −1521 to +41 relative to cpdA IC) This study pSMK-cpdA2 pHK0011 with cpdA upstream region (positions −1178 to −1051 relative to cpdA IC) This study pSMK-cpdA3 pHK0011 with cpdA upstream region (positions −1163 to −1051 relative to cpdA IC) This study pGEM-11zf(+) General cloning vector; Ap r Promega pGEM-11zf(+)-cpdA2mt pGEM-11zf(+) with the mutagenized sequence for the putative CRP-binding site of cpdA regulatory region This study pSMK-cpdA2mt pSMK-cpdA2 but mutagenized CRP-binding site This study Open in a separate window Strains and plasmids used in this study

Techniques: Sequencing, Binding Assay