Bacterial strains and plasmids used in this study are listed in Table S1. C. burnetii Nine Mile phase II (RSA439, clone 4, NMII) genetic transformants were axenically cultured in ACCM-D as previously described (46 (link)). Bacterial stocks were maintained in ACCM-D containing 10% dimethyl sulfoxide and frozen at −80°C. Escherichia coli Stellar (TaKaRa) and E. coli PIR2 (Invitrogen) cells were used for recombinant DNA procedures and cultivated in Luria-Bertani broth or Terrific broth. HeLa (CCL-2; American Type Culture Collection [ATCC]) human cervical epithelial cells were cultured in Dulbecco modified Eagle medium (DMEM) (Thermo Fisher) containing 10% fetal bovine serum (FBS) (Thermo Fisher) at 37°C and 5% CO2. THP-1 macrophages (TIB-202; ATCC) and African green monkey kidney (Vero) cells (CCL-81; ATCC) were maintained in RPMI 1640 medium containing 10% FBS at 37°C and 5% CO2. C. burnetii CRISPRi strains were generated by electroporating C. burnetii Nine Mile phase II grown in ACCM-D with pB-CRISPRi and pTnS2::1169P-tnsABCD plasmids (Table S1) and selecting transformants in ACCM-D lacking proline, as previously described (9 (link), 28 (link)).
E coli pir2
Manufactured by Thermo Fisher Scientific
The E. coli PIR2 is a laboratory strain of Escherichia coli bacteria. It is commonly used as a host for the propagation and maintenance of plasmid DNA in molecular biology and genetic engineering applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
E coli dh5α, by Thermo Fisher Scientific (2 mentions)
Carbenicillin carb, by IBI Scientific (2 mentions)
Sodium acetate, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ccl 81, by American Type Culture Collection (1 mentions)
Hela ccl 2, by American Type Culture Collection (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by GoldBio (1 mentions)
Erythromycin erm, by Merck Group (1 mentions)
Gentamicin gm, by Merck Group (1 mentions)
E coli stellar, by Takara Bio (1 mentions)
Erythromycin em, by Merck Group (1 mentions)
5 protocols using e coli pir2
1
Generation of C. burnetii CRISPRi Strains
2
Anaerobic Bacteroides and E. coli Culturing
3
Cultivation and Storage of C. burnetii
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Bacterial strains used in this study are listed in Table S1 . The C. burnetii Nine Mile phase II (RSA439, clone 4, NMII) strain was used for this work. NMII C. burnetii and genetic transformants were axenically cultured in ACCM‐D as previously described (Sandoz et al., 2014 (link)), with the addition of 4‐hydroxyphenylpyruvic acid (4‐HPP) for tyrosine‐based nutritional selection. For storage, bacteria were pelleted following 7 days of growth, washed three times in phosphate‐buffered saline (PBS; 1 mM KH2PO4, 155 mM NaCl, 3 mM Na2HPO4, pH 7.4), and then suspended in a freezing medium (ACCM‐D containing 10% dimethyl sulfoxide) and frozen at −80°C. E. coli Stellar (Takara) and E. coli PIR2 (Invitrogen) cells were used for recombinant DNA procedures and cultivated in Luria‐Bertani (LB) broth or terrific broth. THP‐1 macrophages (TIB‐202; ATCC) and African green monkey kidney (Vero) cells (CCL‐81; ATCC) were maintained in an RPMI‐1640 medium containing 10% FBS at 37°C and 5% CO2. C. burnetii replication in host cells or in ACCM‐D was measured by quantitative PCR of genome equivalents (GE) as previously described (Howe et al., 2010 (link); Omsland et al., 2009 (link)) using a probe specific to groEL.
4
Gateway Cloning for Fluorescent Tags
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Gateway entry sclones were created for C-terminal tagging with fluorescent proteins by replacing the default 3xHA sequence in plasmid pR6K-attL1-3xHA-hdhfr-yfcu-attL2 (Pfander et al., 2013 (link)) with GFP-mu3 (Addgene plasmid 20410) (Franke-Fayard et al., 2004 (link)), iLov (Addgene plasmid 26769) (Chapman et al., 2008 (link)), mEmerald (a kind gift from J. Liu), mCherry (from RMgmDB plasmid pL0046), and mVenus. Entry clones with R6K origins were maintained in E. coli PIR2 (Invitrogen).
5
Anaerobic Culturing and Genetic Manipulation
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Detailed information of the strains used in this study is provided in Table S1. All anaerobes were cultured in an anaerobic chamber with an atmosphere of 83% N2, 2% H2 and 15% CO2 at 37 ºC.
For most experiments, Bacteroides strains, AC, CC were grown in Anaerobe Basal Broth (ABB, Oxoid), ER was grown in ABB media with the addition of 3.3 mM sodium acetate (Sigma) and RI was grown in Brain Heart Infusion Broth (BHI, Sigma). We used E. coli pir2 (Invitrogen) for cloning and maintenance of plasmids with R6K origin (pNBU2-ermGb derivatives and pFW1000 derivatives, Table S2). E. coli DH5α (Thermo Fisher Scientific) was used for cloning and maintenance of plasmids with p15A, pSC101ts and ColE1 origins (pFW2000 derivatives, pFW3000 and pFW4000). We used E. coli BW29427 (E. coli Genetic Stock Center, CGSC) for E. coli-Bacteroides conjugations. All E. coli strains were grown aerobically in Luria Bertoni (LB) media. To support the growth of E. coli BW29427, we supplemented LB media with 25 nM of 2,6-Diaminopimelic acid (DAP, Sigma). We used the following antibiotics when required including 100 µg mL -1 carbenicillin (Carb, IBI Scientific), 25 µg mL -1 erythromycin (Em, Sigma) and 200 µg mL -1 gentamicin (Gm, Sigma). We used 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) for the induction of FnCpf1.
For most experiments, Bacteroides strains, AC, CC were grown in Anaerobe Basal Broth (ABB, Oxoid), ER was grown in ABB media with the addition of 3.3 mM sodium acetate (Sigma) and RI was grown in Brain Heart Infusion Broth (BHI, Sigma). We used E. coli pir2 (Invitrogen) for cloning and maintenance of plasmids with R6K origin (pNBU2-ermGb derivatives and pFW1000 derivatives, Table S2). E. coli DH5α (Thermo Fisher Scientific) was used for cloning and maintenance of plasmids with p15A, pSC101ts and ColE1 origins (pFW2000 derivatives, pFW3000 and pFW4000). We used E. coli BW29427 (E. coli Genetic Stock Center, CGSC) for E. coli-Bacteroides conjugations. All E. coli strains were grown aerobically in Luria Bertoni (LB) media. To support the growth of E. coli BW29427, we supplemented LB media with 25 nM of 2,6-Diaminopimelic acid (DAP, Sigma). We used the following antibiotics when required including 100 µg mL -1 carbenicillin (Carb, IBI Scientific), 25 µg mL -1 erythromycin (Em, Sigma) and 200 µg mL -1 gentamicin (Gm, Sigma). We used 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) for the induction of FnCpf1.
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