The bacterial strains used in this study were E. coli BL21(DE3) (Novagen) and JM109(DE3) (Promega). Disruption of the cca gene (cca::cam, Δcca) was achieved using the Quick & Easy E. coli gene deletion kit #K006 (Genebridges). E. coli TOP10 or NEB 5-alpha were used for cloning and mutagenesis experiments.
Jm109 de3
Manufactured by Promega
Sourced in United States
JM109(DE3) is a competent E. coli bacterial strain commonly used in molecular biology applications. It is designed for efficient and reliable protein expression. The strain carries the DE3 lysogen, which enables the expression of target proteins under the control of the T7 promoter system.
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Lab products found in correlation
Sodium chloride (nacl), by Merck Group (1 mentions)
Taq dna polymerase, by Transgene (1 mentions)
All in one first strand cdna synthesis supermix for qpcr, by Transgene (1 mentions)
Dna clean up kit, by CWBIO (1 mentions)
Fastpure plasmid mini kit, by Vazyme (1 mentions)
Xbai hf, by New England Biolabs (1 mentions)
Cell rna kit, by Yeasen (1 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
Ecori hf, by New England Biolabs (1 mentions)
T4 ligase enzyme, by New England Biolabs (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
Bl21 de3, by Transgene (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
Hieff trans liposomal transfection reagent, by Yeasen (1 mentions)
Bca protein quantification kit, by Yeasen (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Bl21 star de3, by Thermo Fisher Scientific (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
8 protocols using jm109 de3
1
Bacterial Strain Generation and Modification
2
Recombinant Protein Expression in E. coli
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The E. coli strain JM109(DE3) from Promega (USA) was chosen because it is a well-characterized microorganism and is recommended for protein expression with the pET system [27 (link)]. This strain contains the expression plasmid pFM23 (Figure 2 and Figure S1 of Supplementary Material ), which was previously obtained by cloning the eGFP gene into the pET28a vector [14 (link)]. The overnight cultures, as well as the growth curves (Session 1), were prepared in Luria-Bertani (LB) medium. This commercial medium is composed of 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl (LB-Miller, Merck KGaA, Darmstadt, Germany) and is commonly used for recombinant protein expression with the pET system [16 ]. For maintenance of selective pressure, the antibiotic kanamycin was added to the growth medium at a final concentration of 30 µg/mL.
3
eGFP Expression in E. coli JM109(DE3)
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The E. coli strain JM109(DE3) from Promega (Madison, WI, USA) was transformed with the plasmid pFM23 (constructed from pET28A vector; Novagen, Madison, WI, USA) for the cytoplasmic production of eGFP [24 (link)]. This plasmid contains a kanamycin resistance gene and a pMB1 origin of replication (medium-copy number replicon), and uses the T7 promoter for transcription of the foreign gene, which can be induced by lactose or its non-hydrolyzable analogue isopropyl β-d -1-thiogalactopyranoside (IPTG).
4
Molecular Cloning and Cell Culture Protocol
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High fidelity restriction endonucleases BamHI-HF, HindIII-HF, EcoRI-HF, XbaI-HF, and T4 ligase enzyme were purchased from NEB (New England Biolabs, MA, USA). Bacteria strain DH10B, BL21(DE3), Taq DNA polymerase, and All-in-One First-Strand cDNA Synthesis SuperMix for qPCR were purchased from Transgen, and JM109(DE3) was purchased from Promega (Promega, WI, USA). pT7Oi and pT7Om vectors were already constructed by our group. psilence-2.1-U6-Hygro vector was purchased from Beijing Rambolide Trading Co. DNA fragments were purified with AxyPrep DNA Gel Extraction Kit (Axygen) or DNA Clean-up Kit (Cwbio), and plasmids were extracted by FastPure Plasmid Mini Kit (Vazyme) or Endo-Free Plasmid Mini Kit I (Omega). Cell RNA Kit, BCA protein quantification kit, and Hieff Trans liposomal transfection reagent were purchased from Yeasen. ProtLytic Protein Lysis and Sample Loading was purchased from New Cell & Molecular biotech Co. Human embryonic kidney 293T (HEK293T) was a gift from Professor Yao Shaohua’s laboratory. Michigan Cancer Foundation-7 (MCF-7) and human hepatocellular carcinomas (Hep G2) were purchased from ATCC (American Type Culture Collection, VA, USA).
5
Preparation of E. coli JM109(DE3) Starter Culture
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E. coli JM109(DE3) from Promega (Madison, WI, USA) was selected for this study because it has been used in previous works from our group for the evaluation of initial adhesion in antifouling surfaces [67 (link)] and because it has was shown to have similar biofilm formation behavior to different clinical isolates, including E. coli CECT434 [78 (link)]. A starter cell culture was obtained using the same procedure as described in Moreira, et al. [79 (link)]. In brief, the cells were collected from a cryo-preserved batch (1 mL aliquots in glycerol stock kept at a constant −80 °C) and thawed at room temperature. Then, 200 mL of culture medium, prepared as previously described [80 (link)], were inoculated with 500 μL of cell suspension and incubated overnight at 37 °C with a constant orbital agitation of 120 rpm. A volume of 60 mL of the cultured bacteria was centrifuged (Eppendorf Centrifuge 5810R, Hamburg, Germany) at 3202× g for 10 min, resuspended in citrate buffer at pH 5 and centrifuged again in the same conditions. The final cell suspension was then diluted in citrate buffer until an optical density (OD600 nm) of 0.1, corresponding to a cell density of 7.6 × 107 cells mL−1.
6
Cloning and Expression of TbLysoPLA
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The TbLysoPLA gene had been previously cloned in T.b. brucei phospholipases study context (Monic & al, manuscript in revision). Briefly, the TbLysoPLA open reading frame (ORF) (Tb427.08.6390) was amplified by PCR from T.b. brucei 427 genomic DNA using specific primers: aS (TbLysoPLA-pGEX-Fw): 5’-AGCTCGAGATGTTTGGCACGCCGTTGAGAAT-3’; aAS (TbLysoPLA-pGEX-Rv): 5’-AGGCGGCCGCTTACGATTTCGATGAAGGTCCGG-3’. The 861 bp product was cloned using the restriction enzymes Xho1 and Not1 (sites included in the primer sequences) into the plasmid pGEX-4T-1 (GE Healthcare 28-9545-49) in frame with the GST (Glutathione- S-Transferase) after the thrombin cleavage site. The TbGK (Tb927.9.12630) ORF had been previously cloned in the pET151 plasmid at the University of Tokyo [18 (link)], and was provided by the UMR-5234-MFP laboratory. The recombinant vectors have been used to transform Escherichia coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA, United States) and JM109 (DE3) (Promega, Fitchburg, WI, United States) strains for TbLysoPLA and TbGK expression respectively.
7
Purification of Polyhistidine-Tagged Proteins
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pRSETB containing a gene encoding protein tagged with N-terminal polyhistidine tags was transformed into JM109 (DE3) (Promega, Madison, WI, USA) by heat shock transformation at 42 oC for 45 s. The transformants were then plated onto agar plates containing 0.1 mg/mL carbenicillin. Colonies were cultured in 200 mL LB media containing 0.1 mg/mL carbenicillin at 23 °C with gentle shaking at 80 rpm for 4 days. Polyhistidine-tagged proteins were purified by Ni-NTA agarose (Qiagen, Hilden, Germany) chromatography, then eluted using 200 mM imidazole in TN buffer (10 mM Tris-HCl pH 8, 150 mM NaCl). The eluted proteins were processed with buffer exchange chromatography using a PD-10 column (GE Healthcare, Chicago, IL, USA). The final elution was diluted in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-KOH (pH 7.4).
8
E. coli JM109(DE3) Biofilm Formation
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E. coli JM109(DE3) from Promega (USA) was used in this study because it has shown a good biofilm forming capacity in a diversity of platforms operated at different shear stresses (Gomes et al., 2014; Moreira et al., 2015b; Teodósio et al., 2011) . Additionally, it was shown that its biofilm formation was similar to other E. coli strains which are often used for antimicrobial susceptibility tests (Gomes et al., 2014) . The strain was grown overnight at 30ºC and 120 rpm after the inoculation of 500 µl of a glycerol stock kept at -80ºC in 0.2 l of culture medium (Teodósio et al., 2011) . This medium consisted of 5.5 g l -1 glucose, 2.5 g l -1 peptone, 1.25 g l -1 yeast extract in phosphate buffer (1.88 g l -1 KH2PO4 and 2.60 g l -1 Na2HPO4), pH 7.0. All medium components were purchased from Merck KGaA (Germany).
Cells were then centrifuged (3202 g, 10 min) and washed twice with citrate buffer 0.05 mol l - 1 , pH 5.0 (Moreira et al., 2015b) . The pellet was resuspended in citrate buffer and the cellular suspension was adjusted to a final concentration of 7.6 × 10 7 cell ml -1 determined by optical density at 610 nm (OD610 nm = 0.1). This cell suspension was used for initial biofilm formation assays in 96-well microtiter plates and in a parallel plate flow chamber (PPFC).
Cells were then centrifuged (3202 g, 10 min) and washed twice with citrate buffer 0.05 mol l - 1 , pH 5.0 (Moreira et al., 2015b) . The pellet was resuspended in citrate buffer and the cellular suspension was adjusted to a final concentration of 7.6 × 10 7 cell ml -1 determined by optical density at 610 nm (OD610 nm = 0.1). This cell suspension was used for initial biofilm formation assays in 96-well microtiter plates and in a parallel plate flow chamber (PPFC).
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