Polymerase chain reaction (PCR) was performed using OneTaq (New England Biolabs), Phusion (New England Biolabs), or PfuTurbo (Agilent) as per manufacturer guidelines with oligonucleotides purchased from Integrated DNA Technologies. All mutations or gene fusions were created by overlap extension PCR. Gene sequences from S. typhimurium were PCR amplified from the T3SS-competent strains SB300 (wild-type; gift of J. Galán) or SB1741 (3xFLAG::SpaO, silent SpaO L79CTG to L79CTA variant; gift of J. Galán)4 (link). The T4 lysozyme (C54T, C97A) sequence was obtained from Addgene plasmid 18111. An additional mutation (D20N) in T4 lysozyme was made to decrease toxicity in E. coli21 (link), and the terminal three residues were mutated to alanines to decrease conformational entropy. Standard molecular biology protocols were followed to clone sequences of interest into modified pCDFduet or pETduet vectors for expression in E. coli or pBAD for expression in S. typhimurium. Restriction enzymes and Quick Ligase (New England Biolabs) were used as per manufacturer specifications. Salmonella genomic mutants were produced using homologous recombination from SacB-expressing suicide plasmids22 (link). All SpaO and OrgB mutants were prepared on the SB1741 background. FliM and FliN mutants were prepared on the SB300 background.
Oligonucleotides
Manufactured by Agilent Technologies
Oligonucleotides are short sequences of synthetic DNA or RNA molecules. They are used as probes, primers, and building blocks in various molecular biology applications, such as gene synthesis, DNA sequencing, and diagnostic assays.
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Lab products found in correlation
6 protocols using oligonucleotides
1
Molecular Cloning and Mutagenesis Pipeline
2
Genome-wide Array Profiling of Blood DNA
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DNA was extracted from whole blood by the Puregene DNA Blood Kit (Gentra) according to the manufacturer’s instructions. The procedures for DNA digestion, labeling, and hybridization for the oligo arrays were performed according to the manufacturers’ instructions. Seven of the eight subjects were studied with custom-designed genome-wide array with approximately 180,000 oligonucleotides, manufactured by Agilent Technologies, Inc. (Santa Clara, CA) as previously described [22 (link)]. The clinical array is designed by the MGL at BCM with exon-by-exon coverage for about 1,700 genes and 700 microRNAs. Confirmatory FISH analyses for 17q25 deletion were performed using RP11-497H17 and RP11-1182P23. Subject 3 was studied in the Cytogenetics Laboratory at Cincinnati Children’s Hospital using both bacterial artificial chromosome (BAC) and single nucleotide polymorphism (SNP) arrays. The SignatureSelect V2 chip containing approximately 4671 BAC clones concentrated in areas of clinical significance, was used for array-CGH. Additional analysis was performed using the Infinium Assay with the Illumina HD Human610-quad BeadChip platform containing approximately 620,900 markers.
3
Codon-Optimized Protein Design
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Designs were padded to 65 amino acids with serine. Then, the designs were codon-optimized for expression in Saccharomyces cerevisiae. Oligonucleotides encoding the designs and SSM mutations were purchased from Agilent technologies. Libraries were amplified as previously described42 (link).
4
Multiplex CRISPR Screening of Peptide Substrates
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Sixty-three peptide substrates with well-resolved degron motifs were selected for analysis by multiplex CRISPR screening. The peptides substrates were divided into three pools of equal size based on their stability, synthesized as oligonucleotides (Agilent) and cloned into the GPS vector downstream of GFP. An sgRNA library targeting known Cullin adaptors (259 genes at a depth of 4 sgRNAs per gene) was synthesized (Agilent) and cloned into lentiCRISPR v2 as described above; the U6-sgRNA cassette was then amplified by PCR and cloned into the GPS vector using the I-SceI site to generate the multiplex CRISPR screening library. Screens were performed in the 1-bin format as described above.
5
Molecular Cloning and Genetic Engineering Protocols
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PCR was performed using OneTaq (New England Biolabs), Phusion (New England Biolabs) or PfuTurbo (Agilent) as per manufacturer guidelines with oligonucleotides purchased from Integrated DNA Technologies. All mutations or gene fusions were created by overlap extension PCR. Gene sequences from S. typhimurium were PCR amplified from the T3SS-competent strains SB300 (wild type; gift from J. Galán) or SB1741 (3 × FLAG::SpaO, silent SpaO L79CTG to L79CTA variant; gift from J. Galán)4 (link). The T4 lysozyme (C54T, C97A) sequence was obtained from Addgene plasmid 18111. An additional mutation (D20N) in T4 lysozyme was made to decrease toxicity in E. coli21 (link), and the terminal three residues were mutated to alanines to decrease conformational entropy. Standard molecular biology protocols were followed to clone sequences of interest into modified pCDFduet or pETduet vectors for expression in E. coli or pBAD for expression in S. typhimurium. Restriction enzymes and Quick Ligase (New England Biolabs) were used as per manufacturer specifications. Salmonella genomic mutants were produced using homologous recombination from SacB-expressing suicide plasmids22 (link). All SpaO and OrgB mutants were prepared on the SB1741 background. FliM and FliN mutants were prepared on the SB300 background.
6
Design of Custom CRISPR sgRNA Library
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For the design of the custom sgRNA library targeting essential genes and safehavens we used the Broad GPP sgRNA design portal and the safe-havens as designed previously (15) (link). The sgRNA sequences (Supplemental Table 2) were ordered as a pool of oligonucleotides (Agilent) with flanking sequences to enable PCR amplification and Gibson assembly into pLentiGuide-Puro (pLG, addgene #52963). The pooled oligo library was amplified using pLG_U6_foward 5'-GGCTTTATATATCTTGTGGAAAGGACGAAACACCG-3' and pLG-TRACR_Reverse 5'-GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'. The fragments were purified and cloned into pLG as described by Morgens (16) (link). The representation of the custom sgRNA library was validated by next generation sequencing.
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