A shuttle vector pMM7 was constructed for the targeted deletion of spo0A in P. thermoglucosidasius DSM2542. The fragments were assembled through USER cloning (New England Biolabs, US), as described in previous section. A full list of primers used for the amplification of fragments is shown in Table 2 . Backbone fragments were amplified using the temperature-sensitive vector pGB-sfGFP-best as template, while the homologous arms (of 750 bp each) were amplified from P. thermoglucosidasius DSM2542 gDNA. All fragments were purified using a NucleoSpin Gel and PCR kit (Macherey–Nagel, Germany). The plasmid was assembled through USER cloning, after which they were directly transformed into chemically competent E. coli DH5α-λpir (Table 1 ). After recovery, the transformed cells were plated onto LB agar plates containing 6.25 mg/L kanamycin. Following incubation at 37 °C, colony PCR was performed with primer set 11M2/12 (Table 2 ) in order to identify colonies containing correctly assembled plasmid. The PCRs were performed using OneTaq Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs, US), in accordance with manufacturers’ protocol. Positive colonies were purified using a NucleoSpin Plasmid kit (Macherey–Nagel, Germany), and the correct assembly of the purified plasmid was confirmed through sequencing (Eurofins, Germany) with primers 11M2 and 12.
User cloning
Manufactured by New England Biolabs
Sourced in United States
USER (Uracil-Specific Excision Reagent) cloning is a highly efficient and versatile DNA cloning method. It enables the seamless assembly of DNA fragments without the need for restriction enzymes or ligase. The core function of USER cloning is the creation of compatible DNA overhangs through the controlled excision of uracil residues, allowing for the directional assembly of multiple DNA fragments in a single reaction.
Automatically generated - may contain errors
Lab products found in correlation
Gblocks gene fragment, by Integrated DNA Technologies (3 mentions)
Milli q water purification system, by Merck Group (3 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (3 mentions)
Kld enzyme mix, by New England Biolabs (3 mentions)
Kod hot start dna polymerase, by Merck Group (2 mentions)
Pfuturbo cx hotstart dna polymerase, by Agilent Technologies (2 mentions)
Phusion u hot start dna polymerase, by Thermo Fisher Scientific (2 mentions)
45 60 mer oligonucleotides, by Integrated DNA Technologies (2 mentions)
Neb turbo, by New England Biolabs (2 mentions)
Dh5α cells, by New England Biolabs (2 mentions)
Veraseq ultra dna polymerase, by Qiagen (2 mentions)
Neb 10 beta competent cells, by New England Biolabs (2 mentions)
Maxiprep, by Qiagen (2 mentions)
Neb stable competent e coli, by New England Biolabs (2 mentions)
Spcas9 hairpin u6 sgrna expression plasmid, by Addgene (2 mentions)
Templiphi rolling circle amplification, by Thermo Fisher Scientific (2 mentions)
5 cbh aav abe npuc u6 sgrna, by Addgene (2 mentions)
Aav abe npun, by Addgene (2 mentions)
Mach1 chemically competent e coli, by Thermo Fisher Scientific (2 mentions)
Bbsi plasmid digest, by New England Biolabs (2 mentions)
10 protocols using user cloning
1
Targeted Deletion of spo0A in P. thermoglucosidasius
2
Genetic engineering of Bt Cry1Ac toxin
3
Precise APOBEC1 Variant Generation
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PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Plasmids for BE and sgRNA were constructed using USER cloning (New England Biolabs) from previously reported plasmids1 (link). DNA vector amplification was carried out using NEB 10beta competent cells (New England Biolabs). Site-directed mutagenesis of APOBEC1 variants was done using blunt-end ligation. Briefly, a primer with an overhang containing the desired point mutation was used to amplify the appropriate vector plasmid by PCR. KLD enzyme mix (New England Biolabs) was used to phosphorylate and circularize the PCR product prior to transformation.
4
Precise APOBEC1 Variant Generation
5
Cloning and Truncation of ProTα
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PCR was performed using Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs). Candidate human protein DNAs were purchased from IDT as gBlock Gene Fragments. Bacterial expression plasmids encoding human protein fused to Cre were made using USER-cloning (New England BioLabs). Truncation of ProTα was done using blunt-end ligation to delete regions of ProTα. Following PCR, KLD enzyme mix (New England BioLabs) was used to phosphorylate and circularize the PCR product before transformation into NEB10beta cells (New England BioLabs).
6
Optimized Molecular Cloning Protocol
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All PCR reactions were performed using PfuTurbo Cx polymerase (Agilent Technologies) or VeraSeq ULtra polymerase (Enzymatics). Water was purified using a MilliQ water purification system (Millipore, Billerica, MA). All MPs were constructed using USER cloning (New England Biolabs). Native E. coli genes were amplified by PCR directly from genomic DNA, and non-bacterial genes were synthesized as bacterial codon-optimized gBlocks Gene Fragments (Integrated DNA Technologies). All DNA cloning was carried out using NEB Turbo cells (New England Biolabs).
7
Plasmid Construction and AAV Cloning
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Base editor plasmids were constructed by replacing deaminase and Cas-protein domains of the p2T-CMV-ABE7.10-BlastR (Addgene 152989) plasmid by USER cloning (New England Biolabs)(50 (link)). Individual sgRNAs were cloned into the SpCas9-hairpin U6 sgRNA expression plasmid (Addgene 71485) using BbsI plasmid digest and Gibson assembly (New England Biolabs). Protospacer sequences and gene-specific primers used for amplification followed by HTS are listed in Supplementary Table 1 . Constructs were transformed into Mach1 chemically competent E. coli (ThermoFisher) grown on LB agar plates and liquid cultures were grown in LB broth overnight at 37 °C with 100 μg/mL ampicillin. Individual colonies were validated by Templiphi rolling circle amplification (ThermoFisher) followed by Sanger sequencing. Verified plasmids were prepared by mini, midi, or maxiprep (Qiagen).
AAV vectors were cloned by Gibson assembly (NEB) using NEB Stable Competent E. coli (High Efficiency) to insert the sgRNA sequence and C-terminal base editor half of ABE8e-SpyMac into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half and a second U6-sgRNA cassette into v5 Cbh-AAV-ABE-NpuN (Addgene 137178)(74 (link)).
AAV vectors were cloned by Gibson assembly (NEB) using NEB Stable Competent E. coli (High Efficiency) to insert the sgRNA sequence and C-terminal base editor half of ABE8e-SpyMac into v5 Cbh-AAV-ABE-NpuC+U6-sgRNA (Addgene 137177), and the N-terminal base editor half and a second U6-sgRNA cassette into v5 Cbh-AAV-ABE-NpuN (Addgene 137178)(74 (link)).
8
Plasmid Construction and AAV Cloning
9
Genetic engineering of Bt Cry1Ac toxin
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PCR was performed using PfuTurbo Cx Hotstart DNA polymerase (Agilent Technologies), VeraSeq ULtra DNA polymerase (Enzymatics), or Phusion U Hot Start DNA Polymerase (Life Technologies). Water was purified using a MilliQ water purification system (Millipore). Plasmids and selection phages were constructed using USER cloning (New England Biolabs). Genes were either synthesized as bacterial codon-optimized gBlocks Gene Fragments (Integrated DNA Technologies) or amplified by PCR from native sources. Cry1ac was amplified by PCR from the B. thuringiensis strain Bt_B107284 and cloned into the Bt expression vector pMON101647 using Hot Fusion32 (link) to generate the expression plasmid pMON133051, which served as a template for amplifying Cry1ac fragments for constructing PACE vectors. The toxin-binding region from T. ni cadherin (A1133-T1582, AEA29692.10), referred to as TnCAD-FL, was synthesized using 45–60-mer oligonucleotides (Integrated DNA Technologies) by overlap extension PCR using KOD Hot Start DNA polymerase (EMD Millipore). The synthetic wild-type TnCAD-FL template was used to generate the TnTBR3-FL fragment via site-directed mutagenesis using the QuikChange II kit according to the manufacturers’ instructions. (Agilent Technologies). DNA vector amplification was carried out using NEB Turbo or DH5α cells (New England Biolabs).
10
Yeast Plasmid Construction and Transformation
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Saccharomyces cerevisiae strains (Supplementary file 1 ) are derived from Σ1278b (from Bruno Andre Gournas et al., 2017 (link)) or BY4709; all strains are uracil auxotrophs (Ura3-negative). Plasmids are based on pFB001 (Bianchi et al., 2016 (link)) and constructed by User Cloning (New England Biolabs) in a three-way ligation method, where the plasmid is amplified in three similar sized fragments of which one contains the Gene Of Interest (GOI) and the other two form the full ampR gene when ligation is successful. Plasmid fragments were transformed and ligated in Escherichia coli MC1061 by means of the heat shock procedure. Plasmid assembly and nucleotide sequences were confirmed by DNA sequencing, and plasmids isolated from E. coli were transformed to Saccharomyces cerevisiae using the Li-acetate method (Schiestl and Gietz, 1989 (link)) and selection was based on Ura3 complementation. Positive transformants were re-cultured twice to ensure clonality.
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