An SP1 expression vector (pLXSN-SP1) was expanded in DH5α competent cells and used as a template to create the SP1 mutants using the QuikChange II site-directed mutagenesis kit (Agilent Technologies), according to the manufacturer’s instructions. Briefly, to generate the K19R mutation, mutagenic primers (Eurofin) were designed using the Quick-Change Primer Design Program (Agilent Technologies). The first step of the site-directed mutagenesis involved mutant-strand synthesis by PCR using the mutagenic primers, following which the amplified products were treated with DpnI at 37 °C for 1 h to digest the parental methylated and hemimethylated DNA template. The newly synthesized mutated DNA was transformed into XL1-blue super competent cells (Agilent Technologies) for selection of the mutated SP1 clones.
Xl1 blue supercompetent cells
Manufactured by Agilent Technologies
Sourced in United States
XL1-Blue supercompetent cells are a strain of genetically engineered Escherichia coli (E. coli) bacteria commonly used in molecular biology experiments. They are designed to have enhanced competence, allowing for efficient uptake and transformation of DNA plasmids during genetic engineering procedures.
Automatically generated - may contain errors
Lab products found in correlation
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (3 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Bl21 gold de3 cells, by Agilent Technologies (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
L cysteine, by Merck Group (2 mentions)
Fosfomycin disodium salt, by MP Biomedicals (2 mentions)
Dpni, by New England Biolabs (2 mentions)
Quickchange primer design program, by Agilent Technologies (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
Quikchange protocol, by Agilent Technologies (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Ammonium formate, by Merck Group (1 mentions)
G418 sulfate, by Thermo Fisher Scientific (1 mentions)
Iodine 125, by PerkinElmer (1 mentions)
Formic acid, by Merck Group (1 mentions)
20 protocols using xl1 blue supercompetent cells
1
Site-Directed Mutagenesis of SP1 Expression Vector
2
GUCY2C Expression Vector Construction
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Expression vectors pcDNA3.1(+) containing full GUCY2C cDNA sequence with and without the c.2381A > T variant were constructed by BioMatik (BioMatik, Ontario, Canada). Vectors were treated with NotI and AflII restriction enzymes (New England BioLabs, Ipswich, MA) and inserts were gel purified using the Zymoclean Gel DNA recovery kit (ZymoResearch, Irvine, CA), then ligated into the multicloning site of a pcDNA3.1(+) IRES‐GFP expression vector (Addgene, Watertown, MA). The ligation product was introduced into XL‐1 Blue supercompetent cells (Agilent, Santa Clara, CA), and Sanger sequencing was used to confirm the full GUCY2C insert sequence (Supplementary Table S1 ) .
3
Mutagenesis and Protein Expression of bfrB
4
Site-Directed Mutagenesis of proA Alleles
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Wild-type proA alleles from each strain were changed to introduce a codon for Ala at a position corresponding to E383 in the E. coli enzyme using the QuikChange protocol (Agilent). The products were digested by DpnI, and the plasmids were introduced into XL1-Blue Supercompetent cells (Agilent). The cells were plated on LB agar containing 50 µg/ml ampicillin and grown overnight at 37°C. The introduction of the intended mutation was verified by sequencing purified plasmid DNA.
5
Cloning and Expression of EMV CP Gene
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The sequence of the EMV CP gene (wtEMV; GenBank Accession Number: NC_001480.1 ) was obtained as a product of gene synthesis (plasmid pUC57-EMV; BioCat, Heidelberg, Germany) with flanking restriction sites NdeI and XhoI for further subcloning. The wtEMV CP gene was excised from the helper plasmid with NdeI and XhoI restriction enzymes (Thermo Fisher Scientific, Waltham, MA, USA), followed by DNA fragment purification with the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) and cloning into the NdeI and XhoI sites of the E. coli expression vector pET42a(+) (Novagen, Madison, WI, USA), resulting in the pET42-EMV expression vector. XL1-Blue Super competent cells (Agilent Technologies, Santa Clara, CA, USA) were used for plasmid preparations. Clones with the corresponding insert were selected by restriction analysis by digestion with NdeI and XhoI.
6
Recombinant Protein Expression in CHO Cells
7
Plasmid Amplification and Mutagenesis
8
Synthesis and Purification of Bacterial Cofactor
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Buffer salts were
purchased from
Research Products International Corp. (RPI) and used without further
purification. All crystallization materials were from Hampton Research.
Metals were obtained as their chloride salts from J. T. Baker.l -Cysteine was purchased from Sigma Life Sciences. Fosfomycin
disodium salt was from MP Biomedicals, LLC. BSH was synthesized as
bacillithiol disulfide (BSSB) by the Vanderbilt Chemical Synthesis
Core and reduced to BSH prior to use according to published procedures.19 (link) pET28 (FosBSa) was
from Platinum PCR SuperMix, and custom primers were ordered from Invitrogen
(Carlsbad, CA). Restriction enzymes were from New England Biolabs
(Ipswich, MA). The pET20b(+) vector was from EMB Chemicals, Inc. (Gibbstown,
NJ). XL1-Blue supercompetent cells and BL21-Gold (DE3) cells were
from Agilent Technologies (Santa Clara, CA).
purchased from
Research Products International Corp. (RPI) and used without further
purification. All crystallization materials were from Hampton Research.
Metals were obtained as their chloride salts from J. T. Baker.
disodium salt was from MP Biomedicals, LLC. BSH was synthesized as
bacillithiol disulfide (BSSB) by the Vanderbilt Chemical Synthesis
Core and reduced to BSH prior to use according to published procedures.19 (link) pET28 (FosBSa) was
from Platinum PCR SuperMix, and custom primers were ordered from Invitrogen
(Carlsbad, CA). Restriction enzymes were from New England Biolabs
(Ipswich, MA). The pET20b(+) vector was from EMB Chemicals, Inc. (Gibbstown,
NJ). XL1-Blue supercompetent cells and BL21-Gold (DE3) cells were
from Agilent Technologies (Santa Clara, CA).
9
Cloning Fluorescent Protein-Receptor Constructs
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pHmystik-pRK7 was a gift from M. Aurousseau and D. Bowie (Aurousseau, 2015 ). pHmystik generates a monomeric fluorescent protein that fluoresces in the blue-green spectrum. Purified pHmystik-pRK7 DNA was isolated via Mini Prep (QIAGEN) and confirmed by sequencing. Nhe1 restriction enzyme cloning sites were introduced via site-directed mutagenesis into GluN1 and GluN2A after the fourth amino acid of the mature protein (not including signal peptide). Nhe1 restriction enzyme sites were introduced at the 5′ and 3′ ends of the pHmystik insert in the pRK7 backbone. pHmystik-5′,3′ Nhe1, GluN1-Nhe1, and GluN2A-Nhe1 constructs were digested with Nhe1 in the presence of BSA, dephosphorylated with rSAP (NEB M0371S) and then separated on 0.8% agarose gel (1× TAE). Digested pHmystik and linearized GluN1 and GluN2A were extracted via Zymoclean gel-purification (Zymo D4001S). Purified pHmystik was inserted into the vector, with either GluN1 or GluN2A, using Roche rapid DNA ligation kit (04898117001; Roche). Ligation reaction of pHmystik-GluN1 and pHmystik-GluN2A was transformed into XL1-Blue super-competent cells (Agilent Technologies), and isolated colonies were picked for Mini Prep. Purified DNA of desired constructs were confirmed by DNA sequencing.
10
Purification and Characterization of FosB Enzyme
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