Unless stated otherwise all chemicals were purchased from Fluka (Buchs, Switzerland), Sigma (Steinheim, Germany), Merck (Darmstadt, Germany), VWR (Hannover, Germany), or Carl Roth (Karlsruhe, Germany). Primers were synthesized by Sigma (Steinheim, Germany) and Thermo Fisher Scientific (Waltham, MA, USA). Sequencing was performed by Eurofins MWG Operon (Luxembourg, Luxembourg). E. coli TOP10 strain was obtained from Invitrogen (Carlsbad, CA, USA) and E. coli BL21 (DE3) C41 strain was purchased from Lucigen (Middelton, USA). Plasmids pGAPZ and pPICZα were purchased from Invitrogen (Carlsbad, USA).
E coli strain top10
Manufactured by Thermo Fisher Scientific
Sourced in United States
The E. coli strain TOP10 is a laboratory strain commonly used for DNA cloning and plasmid amplification. It is a non-pathogenic, genetically engineered bacterium with a well-characterized genetic background that supports high-efficiency transformation and plasmid maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (3 mentions)
Macs mrna isolation kit, by Miltenyi Biotec (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Synthetic dna oligonucleotides, by Integrated DNA Technologies (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Ppiczα, by Thermo Fisher Scientific (1 mentions)
As 580, by Anaerobe Systems (1 mentions)
Tla 55 rotor, by Beckman Coulter (1 mentions)
Pvax1, by Thermo Fisher Scientific (1 mentions)
Plasmid giga kit, by Qiagen (1 mentions)
S aureus atcc 25923, by American Type Culture Collection (1 mentions)
P aeruginosa atcc 27853, by American Type Culture Collection (1 mentions)
M9 minimal medium, by Merck Group (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
A9518, by Merck Group (1 mentions)
T8032, by Merck Group (1 mentions)
K3007, by Merck Group (1 mentions)
A3256, by Merck Group (1 mentions)
K4000, by Merck Group (1 mentions)
Phusion flash high fidelity pcr master mix, by Thermo Fisher Scientific (1 mentions)
46 protocols using e coli strain top10
1
Molecular Biology Reagents and Strains
2
Investigating Lcl Collagenous Repeats
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To determine the role that Lcl collagenous repeats have in various biological processes, lcl was PCR amplified using genomic DNA from clinical isolates using primers 1 and 2 (Table 2 ). PCR products from clinical isolates LU1536, LR1063, and LR0347 (Table 1 ) were used to create lcl inserts containing 18, 13, and 11 repeats, respectively. Upon PCR amplification of the LU1536 clinical strain, a lcl variant was spontaneously produced which contained 14 repeats, which was also transformed into the lcl knockout strain. The lcl variant containing 2 repeats was synthesized (Genscript) and cloned using the strategy described below. The resulting PCR products and the vector pBH6119 were then digested with XbaI and SphI to generate compatible ends. The PCR products were then ligated into the XbaI and SphI digested pBH6119 vector and transformed into E. coli TOP10 strain (Invitrogen). Transformants were selected by carbenicillin resistance on LB agar. Single colonies were then picked, cultured in LB broth with 50 μg/ml carbenicillin for plasmid extraction. After verification the plasmid was then transformed into Lp02 and Lp02Δlcl (Table 1 ).
3
Cultivation of Borrelia burgdorferi and Related Bacteria
4
Plasmid Propagation in E. coli
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All Escherichia coli strains were grown in Luria-Bertani (LB) broth and agar (Difco). When appropriate, hygromycin and kanamycin were added at 150 and 50 μg/ml into the medium, respectively. Chemically competent E. coli TOP10 strain (Invitrogen) was used for propagation of all plasmids in this study.
5
Genetic engineering of Sulfolobus islandicus
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The strains and plasmids used in this study are listed in Table S2 (available at FigShare [https://doi.org/10.6084/m9.figshare.8285423 ]). The E. coli Top10 strain (Invitrogen, USA) was used to maintain and propagate plasmid DNA and grown in Luria-Bertani (LB) liquid medium at 37°C. When required, 100 μg/ml ampicillin was supplemented in LB agar plates to select ampicillin-resistant clones. S. islandicus RJW004, the genetic host used in this study, was originated from wild-type isolate S. islandicus M.16.4 (41 (link)), which carried in-frame deletions of pyrEF, lacS, and argD (42 (link)). S. islandicus strains were cultured at 76 to 78°C without shaking in dextrin-tryptone liquid medium (0.2% [wt/vol] dextrin, 0.1% [wt/vol] tryptone, pH 3.3), as described previously (29 (link)). When necessary, 20 μg/ml uracil and/or 50 μg/ml agmatine was added. S. islandicus single colonies were isolated on Gelrite-solidified plates, formulated as described previously (42 (link)), via an overlay cultivation approach (43 (link)). Plates were put into sealed plastic bags and cultivated for 10 to 20 days at 76 to 78°C. Cell growth was monitored by optical density measurements at 600 nm (OD600) with a CO8000 cell density meter (WPA, Cambridge, United Kingdom).
6
Synthesis and Integration of Radiolabeled TtoA Protein
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To synthesize TtoA as a
radioactively labeled substrate for in vitro integration
experiments, an S135-based cell-free
protein synthesis system of E. coli was used. S135
is a cytosolic cell extract that was prepared from the E.
coli Top10 strain (Invitrogen) according to the method
described in ref (24 (link)). It allows the expression of proteins by coupled transcription/translation
from plasmid-encoded genes. In vitro synthesis of
a signal sequence-less version of TtoA from plasmid pET28-HisTtoA6 (link) was performed at 37 °C for 20 min as described
previously.24 (link)TtOmp85-containing
proteoliposomes (100 μL) were prepared from 1.5 μg of
purified TtOmp85 according to the method
described in ref (25 (link)). An aliquot of 20 μL of proteoliposomes was combined with
30 μL of in vitro synthesized TtoA to perform in vitro integration at 37 or 70 °C for 15 min. The
proteoliposomes were pelleted at 186000gmax for 30 min at 4 °C in a Beckman TLA-55
rotor, and the pellet was resuspended in 48 μL of INV buffer
[50 mM triethanolamine acetate (pH 7.5), 250 mM sucrose,
and 1 mM dithiothreitol] and supplemented with 12 μL of 5×
SDS loading buffer.24 (link) One aliquot (30 μL)
was treated at 95 °C for 5 min while being shaken at 14000
rpm, whereas the other aliquot was directly loaded on a 15% SDS–polyacrylamide
gel.
radioactively labeled substrate for in vitro integration
experiments, an S135-based cell-free
protein synthesis system of E. coli was used. S135
is a cytosolic cell extract that was prepared from the E.
coli Top10 strain (Invitrogen) according to the method
described in ref (24 (link)). It allows the expression of proteins by coupled transcription/translation
from plasmid-encoded genes. In vitro synthesis of
a signal sequence-less version of TtoA from plasmid pET28-HisTtoA6 (link) was performed at 37 °C for 20 min as described
previously.24 (link)TtOmp85-containing
proteoliposomes (100 μL) were prepared from 1.5 μg of
purified TtOmp85 according to the method
described in ref (25 (link)). An aliquot of 20 μL of proteoliposomes was combined with
30 μL of in vitro synthesized TtoA to perform in vitro integration at 37 or 70 °C for 15 min. The
proteoliposomes were pelleted at 186000gmax for 30 min at 4 °C in a Beckman TLA-55
rotor, and the pellet was resuspended in 48 μL of INV buffer
[50 mM triethanolamine acetate (pH 7.5), 250 mM sucrose,
and 1 mM dithiothreitol] and supplemented with 12 μL of 5×
SDS loading buffer.24 (link) One aliquot (30 μL)
was treated at 95 °C for 5 min while being shaken at 14000
rpm, whereas the other aliquot was directly loaded on a 15% SDS–polyacrylamide
gel.
7
Cloning and Purification of pA27L and pA27LOPT Plasmids
8
Bacterial Strains for PDI Assay
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Two Gram (−)
bacteria and two Gram-positive bacteria were selected to assay PDI. E. coli Top10 strain is purchased from Invitrogen
Co., P. aeruginosa ATCC 27853, S. aureus ATCC 25923, and B. cereus NRRL B-3711 were obtained from the American Type Culture Collection
(ATCC, Rockville, MD, USA), Northern Regional Research Laboratory
(NRRL, USDA, Peoria, Illinois/USA). The GFP expressing E. coli K12 (pSB1C3 backbone) was received as a courtesy
of Sinem Ulusan. All strains of bacteria were cultured in LB medium
or LB agar plates at 37 °C for overnight (16–18 h) incubation.
All bacterial strains were stored with glycerol stocks at −80
°C.
bacteria and two Gram-positive bacteria were selected to assay PDI. E. coli Top10 strain is purchased from Invitrogen
Co., P. aeruginosa ATCC 27853, S. aureus ATCC 25923, and B. cereus NRRL B-3711 were obtained from the American Type Culture Collection
(ATCC, Rockville, MD, USA), Northern Regional Research Laboratory
(NRRL, USDA, Peoria, Illinois/USA). The GFP expressing E. coli K12 (pSB1C3 backbone) was received as a courtesy
of Sinem Ulusan. All strains of bacteria were cultured in LB medium
or LB agar plates at 37 °C for overnight (16–18 h) incubation.
All bacterial strains were stored with glycerol stocks at −80
°C.
9
Bacterial Cultivation and Selection Protocols
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The Pseudomonas savastanoi strains: pv. phaseolicola NPS3121 wild type [34 (link)], oxyR-mutant, oxyR- complemented mutant (oxyR- C) and P. syringae pv. tomato DC3000 were grown or maintained on King´s B (KB, Sigma–Aldrich®, MO, USA) medium or in M9 minimal medium (Sigma–Aldrich®, MO, USA) at 18 °C or 28 °C. The Escherichia coli strains were routinely grown on Luria–Bertani (LB, Dibico®, EdoMex, Mexico) medium at 37 °C. The E. coli Top 10 strain (Invitrogen, CA, USA) was used in the cloning procedures. The E. coli strain JM103 [35 (link)] was used as an indicator strain in the phaseolotoxin assays. When required, the following antibiotics were added for the P. savastanoi strains and E. coli, respectively: ampicillin (Amp) (300 μg mL−1, 100 μg mL−1), kanamycin (Km) (70 μg mL−1, 50 μg mL−1), gentamicin (Gm) 30 μg mL−1, and rifampin (Rf) 50 μg mL−1.
10
Periplasmic Protein Expression via pLIP6-GN Plasmid
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The E. coli Top10 strain (Invitrogen, Carlsbad, CA) was used in cloning work. The E. coli W3110 strain (American Type Culture Collection, N°. 27325) was applied to the expression of recombinant fusion protein.
The pLIP6-GN plasmid was kindly provided to us by Dr Frédéric Ducancel (Department of ImmunoVirology, CEA centre, Fontenay-aux-Roses, France). This vector presents successively in the open reading frame the AP signal sequence and a SfiI/NotI cloning site located between codons +6 and +7 of the E. coli AP gene, thus enabling the recombinant proteins to be directed to the periplasmic space and folding into their native conformation, after induction of the tac promoter with isopropyl-β-D-thiogalactoside (IPTG) [23 (link)].
The pLIP6-GN plasmid was kindly provided to us by Dr Frédéric Ducancel (Department of ImmunoVirology, CEA centre, Fontenay-aux-Roses, France). This vector presents successively in the open reading frame the AP signal sequence and a SfiI/NotI cloning site located between codons +6 and +7 of the E. coli AP gene, thus enabling the recombinant proteins to be directed to the periplasmic space and folding into their native conformation, after induction of the tac promoter with isopropyl-β-D-thiogalactoside (IPTG) [23 (link)].
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