Bacterial strains and plasmids used in this study are shown in Table 1 . Bacillus and E. coli were grown in lysogeny broth (LB, 10 g peptone, 5 g yeast extract, 5 g NaCl per liter, pH 7.0) at 37°C with aeration. Bacillus subtilis harboring pHY300PLK (4.87 kb, Apr, Tcr) or pHYFSM4 (pHY300PLK with aprEFSM4) was cultivated in LB with tetracycline (Tc, 10 μg/ml). E. coli containing pET-26b(+) (Merck Millipore, Germany, 5.4 kb, Kmr) or pETFSM4 (pET-26b(+) with aprEFSM4) was cultivated in LB with kanamycin (Km, 30 μg/ml).
Pet26b
Manufactured by Merck Group
Sourced in Germany
PET26b is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory applications. The core function of PET26b is to facilitate common laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
10xhis tag, by GenScript (1 mentions)
Mlv votu, by GenScript (1 mentions)
Quickchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Bl21 de3 plyss, by Promega (1 mentions)
Pet26b, by Agilent Technologies (1 mentions)
Bl21 codonplus de3 ril, by Agilent Technologies (1 mentions)
Pet19b, by Merck Group (1 mentions)
9 protocols using pet26b
1
Bacillus and E. coli Cultivation and Plasmid Maintenance
2
Engineered HER2 Affibody Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
By adding HEHEHE to the amino terminus of HER2 affibody ZHER2:V2, a new HER2 affibody (HE)3ZHER2:V2 was designed. Using the affibody gene sequence as a template, pET-26b(+) (purchased from Merck, USA) was used as an expression vector, and it was handed over to Sangon Bio-Company (ShangHai, China) for synthesis. The synthesized plasmid containing the affibody gene was double digested with NcoI and EcoRI (produced from BBI, UK) to verify the length of the synthesized gene; and gene sequencing was performed to verify the sequence of the synthesized gene.
3
Overexpression and Purification of vprSJ4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
vprSJ4 was amplified without its own signal sequence using the following primer pairs, pETvprSJ4F (5’-AGAGGATCC GATGGCTCCGGCTTCT-3’, BamHI site underlined) and pETvprSJ4R (5’-AGACTCGAG CGC GAATTCGTCTTC-3’, XhoI site underlined). The amplified fragment was inserted into pET26b(+) (Merck Millipore, Germany) after being digested with BamHI and XhoI. E. coli BL21 (DE3) competent cells were transformed with the ligation mixture by electroporation (200 Ω, 18 kV/cm) [16 (link)]. E. coli cells harboring pETvprSJ4 were grown in LB (250 ml) containing kanamycin (30 μg/ml) until the OD600 of culture reached 0.8. IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to the final concentration of 1 mM, and the culture was incubated for 20 h at 30°C. After centrifugation at 4,000 ×g for 20 min at 4°C, the cell pellet was resuspended in 5 ml of phosphate-buffered saline (PBS, pH 7.2). Cells were then disrupted by sonication (5 cycles of 1 min sonication and 2 min cooling on ice) using a sonicator (UW 2070, Germany). Disrupted cells were centrifuged at 12,000 ×g for 15 min at 4°C. The supernatant (soluble fraction) and cell pellet (insoluble fraction) were obtained and used for experiments.
4
Cloning and Purification of Mutant Epsilon Protoxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene, etxD, encoding epsilon protoxin (P-Etx) with the H162A mutation (P-Etx-H162A) was subcloned from plasmid pCl0 into the expression vector pET-26b(+) (Merck, Darmstadt, Germany) using NcoI and XhoI restriction sites as described previously [45 (link)], which fused the amino-terminal end of P-Etx-H162A to the PelB leader peptide and the carboxy-terminal end of P-Etx-H162A to a polyhistidine (6 × His) affinity tag. Amino acid numbering corresponds to P-Etx with the 13 amino-terminal residues unless otherwise stated. Wild type recombinant P-Etx was produced and purified under ACDP/ACGM (Advisory Committee on Dangerous Pathogens/Advisory Committee on Genetic Modification) containment level 3 conditions.
5
Recombinant PRRS virus OTU and porcine ISG15 expression
6
Cloning and Characterization of Mutant Epsilon Prototoxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene encoding epsilon prototoxin, etxD, from C. perfringens Type D strain NCTC 8346 was cloned into the expression vector pET-26b(+) (Merck, Darmstadt, Germany) with a N-terminal PelB leader peptide in place of the 13 amino acids N-terminal peptide sequence (residues KEISNTVSNEMSK) and with a C-terminal polyhistidine (6× His) tag as described previously [14] (link).
Mutations Y30A and Y196A (amino acid numbering corresponds to prototoxin without the 13 amino acids N-terminal peptide sequence) were introduced into the gene encoding epsilon prototoxin (P-Etx) using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc. Santa Clara, US) according to the manufacturer's instructions. Recombinant P-Etx with Y30A and Y196A mutations is termed Y30A-Y196A.
Recombinant Y30A-Y196A was expressed, purified and its thermostability assessed as described previously [14] (link).
Mutations Y30A and Y196A (amino acid numbering corresponds to prototoxin without the 13 amino acids N-terminal peptide sequence) were introduced into the gene encoding epsilon prototoxin (P-Etx) using the QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc. Santa Clara, US) according to the manufacturer's instructions. Recombinant P-Etx with Y30A and Y196A mutations is termed Y30A-Y196A.
Recombinant Y30A-Y196A was expressed, purified and its thermostability assessed as described previously [14] (link).
7
Recombinant Expression of MlaC and MlaD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA corresponding to MlaC was previously cloned into a custom plasmid (pBE1203) with an N-terminal hexa-histidine tag followed by a TEV protease cleavage site 21 . MlaD DNA corresponding to its periplasmic domain (residues 23-183) was chemically synthesised (Genscript) and cloned into pET vector, pET26b (Merckmillipore) to contain a C-terminal hexa-histidine tag, which we refer to as MlaD Δtm throughout. Both MlaC and MlaD Δtm plasmids were then used to transform E. coli strain BL21(DE3). Expression was performed by growing overnight cultures in lysogeny broth at 37 o C to an OD 600 = 0.6 whereupon protein expression was induced by the addition of IPTG to a final concentration
8
Engineered α-Synuclein Tyr39Trp Variant
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pT7-7 plasmid containing the coding sequence for wild a-SN was acquired from Addgene (plasmid # 36046) (Addgene, Cambridge, MA, USA) [18] . Using specific primers the a-SN codon 39, encoding a tyrosine residue was change by PCR to the tryptophan codon yielding a-SN Tyr39Trp. Synthetic oligonucleotides (primers) were also used for a-SN wild type and a-SN Tyr39Trp, subcloning into pET26b (Merck KgaA, Darmstadt, Germany) vector between the NdeI and XhoI restriction sites.
9
Cloning and Purification of PqsB and PqsC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pqsB (PA0997) and pqsC (PA0998) genes from Pseudomonas aeruginosa PAO1 were amplified from chromosomal DNA by PCR (primers listed in Table S4). pqsB was cloned into pET26b (Merck Millipore) and pqsC was ligated into pET19m or p10$, [71] both based on the pET19b vector (Merck Millipore). The resulting plasmid pET19m-pqsC produces PqsC with an Nterminal His6 tag followed by a recognition motif for TEV (tobacco etch virus) protease, while p10$-pqsC encodes PqsC with an N-terminal His6-tagged T7 lysozyme removable by human rhinovirus 3C protease. The active site cysteine C129 of PqsC was mutated to alanine (PqsC C129A ) or serine (PqsC C129S ) in pET19mod-pqsC and p10$-pqsC by PCR-based mutagenesis (Table S4).
Expression and purification of (His6-)PqsBC, (His6-)PqsBC C129A and PqsBC C129S Recombinant proteins were produced in E. coli BL21(DE3)pLysS (Promega) or BL21-CodonPlus(DE3)-RIL (Agilent Technologies) co-transformed with pET26b-pqsB and pET19m-pqsC or p10$-pqsC. Purification involved nickel affinity and size exclusion chromatography with or without an intermittent protease cleavage and chromatography step to remove the His6-affinity tag (Table S5). The purified proteins were concentrated to 20 -35 mg mL -1 , flash-cooled in liquid nitrogen and stored at -80 °C.
Expression and purification of (His6-)PqsBC, (His6-)PqsBC C129A and PqsBC C129S Recombinant proteins were produced in E. coli BL21(DE3)pLysS (Promega) or BL21-CodonPlus(DE3)-RIL (Agilent Technologies) co-transformed with pET26b-pqsB and pET19m-pqsC or p10$-pqsC. Purification involved nickel affinity and size exclusion chromatography with or without an intermittent protease cleavage and chromatography step to remove the His6-affinity tag (Table S5). The purified proteins were concentrated to 20 -35 mg mL -1 , flash-cooled in liquid nitrogen and stored at -80 °C.
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!