Streptomyces strain SM14 was isolated from the sponge Haliclona simulans (class Demospongiae, order Haplosclerida, family Chalinidae) which was sampled by SCUBA diving at a depth of 15 m in Kilkieran Bay, Galway, Ireland (N 53°18′56.6′′, W 09°40′08.4′′) as previously described (Kennedy et al., 2009 (link)). The NEB® 5-alpha and the BL21(DE3) competent E. coli cells were obtained from New England Biolabs, Inc., United States.
Bl21 de3 competent e coli
Manufactured by New England Biolabs
Sourced in United States, Germany
BL21(DE3) competent E. coli is a strain of Escherichia coli used for protein expression. It is a common bacterial host for recombinant protein production.
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Lab products found in correlation
Neb 5 alpha, by New England Biolabs (1 mentions)
Glutathione magnetic beads, by Thermo Fisher Scientific (1 mentions)
Fugene 6, by Promega (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
Pet28a, by Merck Group (1 mentions)
Ni nta superflow column, by Qiagen (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Goat anti rabbit irdye 800, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Hgf 294 hg, by R&D Systems (1 mentions)
Hnrnpa0, by Cell Signaling Technology (1 mentions)
β actin 13e5, by Cell Signaling Technology (1 mentions)
Ras 27h5, by Cell Signaling Technology (1 mentions)
E cadherin 24e10, by Cell Signaling Technology (1 mentions)
13 protocols using bl21 de3 competent e coli
1
Isolation of Streptomyces Strain SM14 from Sponge
2
Assay of Cdc42 Activity in Cell Lysates
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The assay of Cdc42 activity in cell lysates was performed as described in ref. 51 (link). Briefly, the p21-binding domain (PBD) domain from PAK1 was subcloned into pGEX-4T1 vector and expressed as an N-terminal GST fusion protein in BL21(DE3) competent E. coli (New England BioLabs). GST-PDB was purified from bacterial lysates using glutathione magnetic beads (ThermoScientific). The PA-Cdc42 lit state mutant (I531E/I539E) or dark-state mutant (C450A, L514K, G528A, L531E, N538E) were expressed in HEK293 cells via transient transfection with Fugene6 (Promega). HEK293 cell line was from Genetica, MA, USA, and it was not authenticated. This cell line has been regularly tested negative for Mycoplasma contamination. Lysates from HEK293 cells were incubated with ~10 μg of GST-PDB beads and incubated at 4 °C for 1 h. Following incubation, beads were washed, boiled with sample buffer, and analyzed via western blot.
3
EHEC Virulence Factors Expression
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EHEC (EDL933) genomic DNA was isolated via a Qiagen DNeasy blood and tissue kit according to the manufacturer’s directions. Sequences encoding LomW (GI:12514345), EscC (GI:12518466), LpfA1 (GI:12518278), and LpfA2 (GI:12518581) were amplified via Phusion polymerase (New England BioLabs) and cloned into a pET30a(+) expression vector using NdeI and XhoI or HindIII-HF (New England BioLabs) restriction sites. The open reading frame encoding each protein was inserted in-frame with a 6×-histidine (His) tag on the C terminus. Ligation, transformation, and expression were performed according to the manufacturer’s directions (pET System; Novagen), with some modifications. Upon confirmation of successful gene insertion via gel electrophoresis and directional sequencing (UTMB Genomics Core), plasmids were transformed into BL21(DE3) competent E. coli (New England BioLabs) via heat shock treatment.
4
Acquiring BL21(DE3) Competent E.coli
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BL21(DE3) Competent E.coli were acquired from New England BioLabs. Details regarding culture conditions were reported previously (Larsen et al., 2018 (link)).
5
Recombinant TTR Protein Expression in E. coli
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BL21 (DE3) competent E. coli (New England BioLabs, Ref #C2527H) were transformed with the pMMHa vector encoding the WT TTR gene. The transformed cells were selected by growing them on agar plates containing 100 µg/mL ampicillin at 37 °C. E. coli starter cultures were initiated by taking a single colony from the agar plate and culturing it in 50 mL LB media in a 250 mL shake flask containing 100 µg/mL ampicillin at 37 °C. These cultures were grown at 37 °C with shaking until bacterial growth indicated by turbidity in the media were observed. The expression cultures were prepared by inoculating 1 L shake flask containing 100 µg/mL ampicillin with a 1:20 dilution of the starter cultures. The expression cultures were grown at 37 °C until OD600 were approximately 0.5, at which point they were induced with 1mM IPTG and incubated overnight at 30 °C with shaking. These cultures were then harvested by centrifugation at 6,000 rpm for 30 minutes at 4 °C. The supernatants were removed, and the pellets were stored at –80 °C until ready for subsequent purification steps.
6
Recombinant Protein Production of BCO Genes
7
Recombinant His-tagged p15 and p16 Purification
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Histidine (His)-tagged p15 and -p16 were amplified using pTRE2pur-p15-Myc and pTRE2pur-p16-Myc as templates, respectively, by PCR using Q5 High-Fidelity DNA Polymerase (New England BioLabs). The primers for His-tagged p15 and His-tagged p16 are listed in Supplementary Table 2 . The PCR products were cloned into pET28a (Millipore) and transformed to into BL21(DE3) competent E. coli (New England BioLabs) to produce recombinant His-tagged p15 and His-tagged p16 after the induction by IPTG. The His-tagged proteins were purified on Ni-NTA Superflow Columns (Qiagen).
8
Recombinant Protein Expression and Analysis
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Plasmid DNA samples with pET20b-short, pET20b-long and empty pET20b vector were transformed separately into BL21(DE3) competent E.coli (New England Biolabs, Germany). The bacterial culture was subsequently induced for gene expression using IPTG (isopropyl b-D-1-thiogalactopyranoside) to a final concentration of 1 mM for 4 h at 37°C. The cells were harvested by centrifugation at room temperature, resuspended in 1X SDS loading dye, denatured by boiling for 3 minutes and centrifuged to remove any insoluble materials. Expression of recombinant protein was analyzed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS—PAGE) and the protein bands were visualized with Coomassie staining solution. The approximate molecular weight was estimated by using prestained protein molecular weight standards.
The protein SDS-gels were electro-blotted onto PVDF membrane (Pierce, USA) using 64 mAmp (0.8mAmp/cm2 of gel) for 90 min. Membrane was blocked using 5% nonfat dry milk in TBS and 0.5% (v/v) Tween-20. Recombinant protein was detected with rabbit antihuman-MTAP (Cell Signaling, cat: 4158S; 1:1000 dilution) as primary antibody and goat anti-rabbit IR Dye 800 (1:3000 dilution; Li-Cor Biosciences) as a secondary antibody. Blots were scanned with Odyssey Infrared Imaging system (Li-Cor Biosciences, Germany), using the following conditions — Channel 700 and signal intensity of 5.
The protein SDS-gels were electro-blotted onto PVDF membrane (Pierce, USA) using 64 mAmp (0.8mAmp/cm2 of gel) for 90 min. Membrane was blocked using 5% nonfat dry milk in TBS and 0.5% (v/v) Tween-20. Recombinant protein was detected with rabbit antihuman-MTAP (Cell Signaling, cat: 4158S; 1:1000 dilution) as primary antibody and goat anti-rabbit IR Dye 800 (1:3000 dilution; Li-Cor Biosciences) as a secondary antibody. Blots were scanned with Odyssey Infrared Imaging system (Li-Cor Biosciences, Germany), using the following conditions — Channel 700 and signal intensity of 5.
9
Overexpression and Purification of Recombinant TTR
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BL21 (DE3) competent E. coli (New England BioLabs, Ref #C2527H) were transformed with the pET-29a vector encoding the N-terminal dual-FLAG-tagged WT TTR gene. The transformed cells were selected by growing them on agar plates containing 50 µg/mL kanamycin at 37 °C. E. coli starter cultures were initiated by taking a single colony from the agar plate and culturing it in 50 mL LB media in a 250 mL shake flask containing 50 µg/mL kanamycin. These cultures were grown at 37 °C with shaking until bacterial growth indicated by turbidity in the media were observed. The expression cultures were prepared by inoculating a 1 L shake flask containing 50 µg/mL kanamycin with a 1:20 dilution of the starter cultures. The expression cultures were grown at 37 °C until OD600 were approximately 0.5, at which point they were induced with 1mM IPTG and incubated overnight at 30 °C with shaking. These cultures were then harvested by centrifugation at 6,000 rpm for 30 minutes at 4 °C. The supernatants were removed, and the pellets were stored at –80 °C until ready for subsequent purification steps.
10
Growth Factor Signaling Pathway Analyses
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Recombinant human fibroblast growth factor 9 (FGF9; #273-F9-025), hepatocyte growth factor (HGF; 294-HG), transforming growth factor alpha (TGFα; 239-A) and epidermal growth factor (EGF; 236-EG) were purchased from R&D systems. Antibodies raised against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), p44/42 MAPK (ERK1/2) (#9102), α-Tubulin (11H10) (#2125), phospho-EGF receptor (Tyr1068) (D7A5) (#3777), EGF receptor (D38B1) XP® rabbit mAb 1:1000 (#4267), GRB2 rabbit antibody (#3972), E-Cadherin (24E10) (#3195), β-Actin (13E5) (#4970), hnRNP A0 (#4046), RAS (27H5) (#3339), and GST (26H1) (#2624), (#7076) were purchased from Cell Signalling, as was an anti-rabbit IgG HRP-linked antibody (#7074). An anti-RFP rabbit antibody (ab62341) was purchased from Abcam and an anti-SOS1 mouse antibody (WH0006654M1) purchased from Sigma-Aldrich. DH5α competent and One Shot™ E. coli were purchased from Thermo Fisher Scientific. BL21 (DE3) competent E. Coli were purchased from NEB.
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