While no experimental model organism was used in the study, we used commercially available E. coli strains for cloning procedures and recombinant protein production. E. coli NEB5α (New England BioLabs) was used for cloning procedures. E. coli BL21 (DE3) (New England BioLabs) and E. coli Rosetta2 (DE3) (Sigma-Aldrich) were used for the recombinant protein production.
E coli rosetta 2 de3
Manufactured by Merck Group
Sourced in Germany
The E. coli Rosetta 2 (DE3) is a bacterial strain used in molecular biology and biochemistry laboratories. It is designed to enhance the expression of eukaryotic proteins in E. coli by providing tRNAs for rare codons found in eukaryotic genes. This strain is commonly used for the production of recombinant proteins.
Automatically generated - may contain errors
Lab products found in correlation
Kta start system, by GE Healthcare (2 mentions)
Hitrap talon crude column, by GE Healthcare (2 mentions)
E coli neb5α, by New England Biolabs (1 mentions)
E coli bl21 de3, by New England Biolabs (1 mentions)
Pet101 d topo vector, by Thermo Fisher Scientific (1 mentions)
Pet28a vector, by Merck Group (1 mentions)
8 protocols using e coli rosetta 2 de3
1
Bacterial Cloning and Protein Production
2
Recombinant K48-linked Polyubiquitin Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human mUbe1 (residues 1–924, mouse E1 enzyme, pET28a, His6-tagged, Thrombin protease site) and Ubc7 (residues 1–165, K48 E2 enzyme, pGEX6P1, GST-tagged, PreScission protease site) enzymes for the K48-linked poly-ubiquitin synthesis were transformed into E. coli Rosetta2 (DE3) (Sigma-Aldrich, Germany) and expressed in TB-media with 1 mM IPTG for 20 h at 18 °C. His6-mUbe1 was purified by Ni-NTA in tandem with an anion exchange chromatography (Q HP column) in 50 mM HEPES, 150 mM KCl, 2 mM MgCl2, pH 7.5. The protein was eluted from the Ni-NTA column with Imidazole followed by washing with the base buffer. The protein was subsequently eluted from the anion exchange column with a KCl gradient. GST-Ubc7 was purified with GSH affinity chromatography in 50 mM HEPES, 150 mM KCl, 10 mM MgCl2, pH 8.0 followed by a gel filtration (Superdex 75 pg 26/60).
3
Overexpression of S. berlinensis ros Genes in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The S. berlinensis ros genes were overexpressed in E. coli Rosetta 2 (DE3) (Merck KGaA, Darmstadt, Germany) using the expression plasmid pET24a(+). Three primer pairs (Table S9 ) introduced restriction endonuclease sites (NdeI and HindIII) for cloning. E. coli Rosetta 2 (DE3) was transformed using a standard protocol for transformation of chemically competent E. coli cells (Sambrook et al., 1989 ). For expression, the resulting strains were aerobically grown at 30°C in 100 mL LB (10 g/L NaCl, 10 g/L tryptone and 5 g/L yeast extract). Ampicillin (100 mg/L) was added when necessary. Expression was induced by adding 0.4 mM isopropylthiogalactopyranoside (IPTG) at an optical density (OD600) of 0.6. After 5 h of further aerobic incubation, cells were harvested by centrifugation (8000 g, 15 min, 4°C). The cell pellets were washed twice with sterile water and stored at −20°C until cell disruption.
4
Purification of Recombinant AdpA Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For details on the protein purification protocol, please see the supplemental material. Briefly, the AdpA proteins of S. venezuelae and S. coelicolor were purified as recombinant C-terminally His-tagged AdpASv (3×Flag-AdpA-6×His; also called AdpASv_His) and C-terminally His-tagged AdpASc (AdpASc_His) (33 (link)), respectively, using the E. coli Rosetta 2(DE3) (Merck) strain harboring the pET28a-3×FLAG-adpA_Sven plasmid (for plasmid construction details, see the supplemental material). Each protein was isolated from a 1.6-L culture conducted in Terrific broth (TB) using affinity chromatography performed using a HiTrap Talon crude column (1 mL, GE Healthcare) and an Äkta start system (GE Healthcare). The protein was eluted using a gradient of imidazole in lysis buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole [pH 8.0]), according to the Äkta system built-in column protocol. The collected proteins were stored at –80°C.
5
Cloning and Purification of PtrCSE1 and PtrCSE2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full‐length coding regions of PtrCSE1 (Potri.003G059200) and PtrCSE2 (Potri.001G175000) were amplified from cDNA of P. trichocarpa (Nisqually‐1) stem differentiating xylem and verified by Sanger sequencing. PtrCSE1 and PtrCSE2 coding regions were then cloned into the pET101/D‐TOPO vector (Invitrogen, Carlsbad, CA) to express recombinant proteins fused with C‐terminal 6×histidine tag. The assembled constructs (PtrCSE1‐pET101 and PtrCSE2‐pET101) were transformed into E. coli Rosetta 2 (DE3) (EMD Millipore Corp., Billerica, MA), and induced for protein expression using 0.2 mm isopropyl β‐d ‐1‐thiogalactopyranoside (IPTG) at 22 °C for 16 h. Recombinant proteins were purified using the Probond Purification Kit (Invitrogen, Carlsbad, CA) as described previously (Shuford et al., 2012 (link)) and stored at −80 °C before enzyme kinetic analysis.
6
Purification of Recombinant AdpA Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For details on the protein purification protocol, please see the supplemental material. Briefly, the AdpA proteins of S. venezuelae and S. coelicolor were purified as recombinant C-terminally His-tagged AdpASv (3×Flag-AdpA-6×His; also called AdpASv_His) and C-terminally His-tagged AdpASc (AdpASc_His) (33 (link)), respectively, using the E. coli Rosetta 2(DE3) (Merck) strain harboring the pET28a-3×FLAG-adpA_Sven plasmid (for plasmid construction details, see the supplemental material). Each protein was isolated from a 1.6-L culture conducted in Terrific broth (TB) using affinity chromatography performed using a HiTrap Talon crude column (1 mL, GE Healthcare) and an Äkta start system (GE Healthcare). The protein was eluted using a gradient of imidazole in lysis buffer A (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole [pH 8.0]), according to the Äkta system built-in column protocol. The collected proteins were stored at –80°C.
7
Genetic Engineering of E. coli and S. cerevisiae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E. coli Rosetta 2 (DE3) and E. coli EPI300 were purchased from Merck Millipore and Epicentre, respectively. S. cerevisiae BJ5464-NpgA (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1 pep4::HIS3 prb1Δ1.6R can1 GAL) was used as the host for homologous recombination. Fungal strains used in this study are Neosartorya fischeri and Pestalotiopsis fici. Plasmids are listed in Table 1 and oligonucleotide sequences are shown in Table S1 . pX330 vector [23 (link), 24 (link)] was used to obtain gene sequence of Cas9 and used as target cleavage plasmid. pET-28a(+) vector was used to produce Cas9 protein. pCC1BAC vector was the starting vector of shuttle plasmids constructed in this study. Another target plasmid pYPZ37 (28.0 kb) harboring Pfma gene cluster was constructed in the previous study [25 (link)].
8
Heterologous expression of FeF3G6″RhaT in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coding regions of FeF3G6″RhaT were subcloned into the pET28a(+) vector (Merck, Darmstadt, Germany) at Nde I and Xho I restriction sites. The resulting plasmids (pET-FeRhaT) were introduced into E. coli Rosetta™ 2(DE3) (Merck). The recombinant FeF3G6″RhaT was expressed and extracted according to the manufacturer's instructions. The recombinant proteins were purified using a His-GraviTrap and concentrated using Amicon Ultra-15.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!