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template fragments  (New England Biolabs)


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    New England Biolabs template fragments
    Template Fragments, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A) Schematic of a simplified protocol to create transposon insertion libraries in Bacteroidales species. Following overnight conjugation on mGAM plates in aerobic conditions, ∼1.5 million transconjugants are inoculated in liquid media (simplified method that improves handling) or plated on more than 25 large petri dishes (solid/conventional method). After 25–28 generations of outgrowth, the liquid culture or colonies scraped from all plates are used for cryostocking and sequencing library preparation, which involves <t>DNA</t> extraction followed by two sequential PCRs to generate amplicons ready for sequencing. Insertion and barcode mapping are done using TnSeeker (Methods). B) Dense and unbiased transposon insertions into the genome of P. merdae , B. uniformis and P. vulgatus type strains. Transposon insertion coverage across the genome is shown as the sum of insertion reads per 10 kbp. Genome size, number of unique insertion positions (Ins) and unique barcodes (BC) are indicated above each plot. C) Most non-essential genes are highly saturated with insertions in all three species, independent of whether the conventional solid or simplified liquid protocol was used. Genes are binned based on the insertion saturation level of TA dinucleotide sites which is the number of TA sites with insertions as a percentage of all TA sites within a gene. D) Solid- and liquid-based libraries show no directionality bias of insertions in the sense (coding) or antisense strand of the targeted gene. E) Mutant abundance is similar in solid- and liquid-generated libraries. Shown is the mean read count of all mutants per gene. One count was added to the reads to enable visualization on a log scale. n = number of genes analyzed, r = Pearson correlation coefficient, p = p -value (two-sided).
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    A) Schematic of a simplified protocol to create transposon insertion libraries in Bacteroidales species. Following overnight conjugation on mGAM plates in aerobic conditions, ∼1.5 million transconjugants are inoculated in liquid media (simplified method that improves handling) or plated on more than 25 large petri dishes (solid/conventional method). After 25–28 generations of outgrowth, the liquid culture or colonies scraped from all plates are used for cryostocking and sequencing library preparation, which involves <t>DNA</t> extraction followed by two sequential PCRs to generate amplicons ready for sequencing. Insertion and barcode mapping are done using TnSeeker (Methods). B) Dense and unbiased transposon insertions into the genome of P. merdae , B. uniformis and P. vulgatus type strains. Transposon insertion coverage across the genome is shown as the sum of insertion reads per 10 kbp. Genome size, number of unique insertion positions (Ins) and unique barcodes (BC) are indicated above each plot. C) Most non-essential genes are highly saturated with insertions in all three species, independent of whether the conventional solid or simplified liquid protocol was used. Genes are binned based on the insertion saturation level of TA dinucleotide sites which is the number of TA sites with insertions as a percentage of all TA sites within a gene. D) Solid- and liquid-based libraries show no directionality bias of insertions in the sense (coding) or antisense strand of the targeted gene. E) Mutant abundance is similar in solid- and liquid-generated libraries. Shown is the mean read count of all mutants per gene. One count was added to the reads to enable visualization on a log scale. n = number of genes analyzed, r = Pearson correlation coefficient, p = p -value (two-sided).
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    A) Schematic of a simplified protocol to create transposon insertion libraries in Bacteroidales species. Following overnight conjugation on mGAM plates in aerobic conditions, ∼1.5 million transconjugants are inoculated in liquid media (simplified method that improves handling) or plated on more than 25 large petri dishes (solid/conventional method). After 25–28 generations of outgrowth, the liquid culture or colonies scraped from all plates are used for cryostocking and sequencing library preparation, which involves <t>DNA</t> extraction followed by two sequential PCRs to generate amplicons ready for sequencing. Insertion and barcode mapping are done using TnSeeker (Methods). B) Dense and unbiased transposon insertions into the genome of P. merdae , B. uniformis and P. vulgatus type strains. Transposon insertion coverage across the genome is shown as the sum of insertion reads per 10 kbp. Genome size, number of unique insertion positions (Ins) and unique barcodes (BC) are indicated above each plot. C) Most non-essential genes are highly saturated with insertions in all three species, independent of whether the conventional solid or simplified liquid protocol was used. Genes are binned based on the insertion saturation level of TA dinucleotide sites which is the number of TA sites with insertions as a percentage of all TA sites within a gene. D) Solid- and liquid-based libraries show no directionality bias of insertions in the sense (coding) or antisense strand of the targeted gene. E) Mutant abundance is similar in solid- and liquid-generated libraries. Shown is the mean read count of all mutants per gene. One count was added to the reads to enable visualization on a log scale. n = number of genes analyzed, r = Pearson correlation coefficient, p = p -value (two-sided).
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    A) Schematic of a simplified protocol to create transposon insertion libraries in Bacteroidales species. Following overnight conjugation on mGAM plates in aerobic conditions, ∼1.5 million transconjugants are inoculated in liquid media (simplified method that improves handling) or plated on more than 25 large petri dishes (solid/conventional method). After 25–28 generations of outgrowth, the liquid culture or colonies scraped from all plates are used for cryostocking and sequencing library preparation, which involves DNA extraction followed by two sequential PCRs to generate amplicons ready for sequencing. Insertion and barcode mapping are done using TnSeeker (Methods). B) Dense and unbiased transposon insertions into the genome of P. merdae , B. uniformis and P. vulgatus type strains. Transposon insertion coverage across the genome is shown as the sum of insertion reads per 10 kbp. Genome size, number of unique insertion positions (Ins) and unique barcodes (BC) are indicated above each plot. C) Most non-essential genes are highly saturated with insertions in all three species, independent of whether the conventional solid or simplified liquid protocol was used. Genes are binned based on the insertion saturation level of TA dinucleotide sites which is the number of TA sites with insertions as a percentage of all TA sites within a gene. D) Solid- and liquid-based libraries show no directionality bias of insertions in the sense (coding) or antisense strand of the targeted gene. E) Mutant abundance is similar in solid- and liquid-generated libraries. Shown is the mean read count of all mutants per gene. One count was added to the reads to enable visualization on a log scale. n = number of genes analyzed, r = Pearson correlation coefficient, p = p -value (two-sided).

    Journal: bioRxiv

    Article Title: A toolkit for transposon libraries and functional genomics in intestinal Bacteroidales

    doi: 10.1101/2025.10.10.681549

    Figure Lengend Snippet: A) Schematic of a simplified protocol to create transposon insertion libraries in Bacteroidales species. Following overnight conjugation on mGAM plates in aerobic conditions, ∼1.5 million transconjugants are inoculated in liquid media (simplified method that improves handling) or plated on more than 25 large petri dishes (solid/conventional method). After 25–28 generations of outgrowth, the liquid culture or colonies scraped from all plates are used for cryostocking and sequencing library preparation, which involves DNA extraction followed by two sequential PCRs to generate amplicons ready for sequencing. Insertion and barcode mapping are done using TnSeeker (Methods). B) Dense and unbiased transposon insertions into the genome of P. merdae , B. uniformis and P. vulgatus type strains. Transposon insertion coverage across the genome is shown as the sum of insertion reads per 10 kbp. Genome size, number of unique insertion positions (Ins) and unique barcodes (BC) are indicated above each plot. C) Most non-essential genes are highly saturated with insertions in all three species, independent of whether the conventional solid or simplified liquid protocol was used. Genes are binned based on the insertion saturation level of TA dinucleotide sites which is the number of TA sites with insertions as a percentage of all TA sites within a gene. D) Solid- and liquid-based libraries show no directionality bias of insertions in the sense (coding) or antisense strand of the targeted gene. E) Mutant abundance is similar in solid- and liquid-generated libraries. Shown is the mean read count of all mutants per gene. One count was added to the reads to enable visualization on a log scale. n = number of genes analyzed, r = Pearson correlation coefficient, p = p -value (two-sided).

    Article Snippet: The transposon vector pCV006 ( ermG ) was constructed using Gibson assembly of different template DNA fragments: pSAM_bt (served as vector backbone, Addgene; 112497), cepA hybrid promoter sequence RBF-103 (synthesized, Eurofins), P BfP1E6 promoter sequence (taken from pWW3864 , kindly provided by Justin Sonnenburg) and a barcode entry sequence containing two BsmBI recognition sequences (synthesized, Eurofins).

    Techniques: Conjugation Assay, Sequencing, DNA Extraction, Mutagenesis, Generated