Journal: eLife

Article Title: Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor

doi: 10.7554/eLife.97437

Figure Lengend Snippet: L. mexicana pTB107 cells expressing tdTomato were co-transfected with Cas12a crRNA-4 template DNA constructs and a cytosine base editor (CBE) single-guide RNA (sgRNA) expression cassette containing a PF16 targeting guide sequence. Isolated DNA from non-clonal mutant populations were subjected to ONT and Illumina sequencing 16 days post transfection. ( A ) Illumina sequencing reads were mapped against the L. mexicana MHOMGT2001U1103 genome annotation. The edited consensus sequence is shown alongside with the reference sequence and calculated editing rates (based on 35× genome coverage). ( B and C ) ONT reads were mapped against two customized L. mexicana MHOMGT2001U1103 genome annotations and viewed in the IGV genome browser , with uniquely mapped reads in gray and reads with multiple alignments in white. Reads mapped against the PGKB 5’ and 3’UTR are over-represented as these UTRs are also present in the pTB107 plasmid. ( B ) ONT reads were mapped to a customized genome in which we assumed that the 18S rRNA SSU locus had one integrated copy of the tdTomato expression cassette on one allele and one integrated copy of the CBE sgRNA expression construct on the other allele. Schematics show the annotation of genetic elements assumed to be on each allele. The position of one allele in respect to the position on the other allele is indicated with red arrows. The position of the Cas12a crRNA-4 to integrate the CBE sgRNA expression cassette is additionally highlighted. Black arrows indicate unique ONT reads that cover the entire customized locus. Reads are trimmed as the genome annotation is incomplete (NNN gap highlighted). ( C ) ONT reads were mapped to a customized genome in which we assumed that both cassettes were integrated on the same allele and could be detected on a single ONT read. ( D ) Illumina read coverage was normalized and chromosome coverage was compared to coverage of individual genetic features on these chromosomes.

Article Snippet: Given the highly repetitive nature of the 18S rRNA SSU locus, we employed Oxford Nanopore Technology (ONT) sequencing to determine the exact integration pattern of both cassettes.

Techniques: Expressing, Transfection, Construct, Sequencing, Isolation, Mutagenesis, Illumina Sequencing, Plasmid Preparation

Journal:

Article Title: DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

doi: 10.1073/pnas.171178898

Figure Lengend Snippet: Strains used in DNA:DNA microarray hybridization analysis

Article Snippet: DNA was typically prepared from 2 ml of exponentially growing culture by using a standard method for bacterial genomic DNA (gDNA) preparation and quantification ( 12 , 13 ). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Bacterium Culture collection Strain origin SSU rRNA accession no. gyrB accession no. S. oneidensis MR-1 ATCC 700550 TS Lake Oneida, NY, sediments AF005251 AF005694 S. oneidensis DLM7 Lower Green Bay sediments, Lake Michigan, Michigan AF387347 * AF005697 S. oneidenisis MR-4 Black Sea water column AF005252 AF005695 Shewanella sp. CL 256/73 NCTC 12093 Human cerebrospinal fluid AF387346 * AF378351 * S. putrefaciens str.

Techniques: Microarray, Hybridization

Journal:

Article Title: DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

doi: 10.1073/pnas.171178898

Figure Lengend Snippet: Phylogenetic tree showing strains of Shewanella based on (A) SSU rRNA and (B) gyrB nucleotide sequence. Relationships calculated by using maximum-likelihood analysis. Species in bold were used in this study. Numbers at nodes represent bootstrap values (of 100 replicates).

Article Snippet: DNA was typically prepared from 2 ml of exponentially growing culture by using a standard method for bacterial genomic DNA (gDNA) preparation and quantification ( 12 , 13 ). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Bacterium Culture collection Strain origin SSU rRNA accession no. gyrB accession no. S. oneidensis MR-1 ATCC 700550 TS Lake Oneida, NY, sediments AF005251 AF005694 S. oneidensis DLM7 Lower Green Bay sediments, Lake Michigan, Michigan AF387347 * AF005697 S. oneidenisis MR-4 Black Sea water column AF005252 AF005695 Shewanella sp. CL 256/73 NCTC 12093 Human cerebrospinal fluid AF387346 * AF378351 * S. putrefaciens str.

Techniques: Sequencing

Journal:

Article Title: DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

doi: 10.1073/pnas.171178898

Figure Lengend Snippet: Relationship between percent similarity in nucleotide sequences for the SSU rRNA (circles) and gyrB (triangles) genes (calculated as percent similarity of test strain relative to the S. oneidensis, MR-1 strain) and percent similarity (31) determined for microarray hybridization profiles between test strains and S. oneidensis, MR-1. Note that the data were not included for E. coli and for env. isolate PV-4, the two most distantly related organisms tested in this study, as the relationship falls off at greater distances.

Article Snippet: DNA was typically prepared from 2 ml of exponentially growing culture by using a standard method for bacterial genomic DNA (gDNA) preparation and quantification ( 12 , 13 ). table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Bacterium Culture collection Strain origin SSU rRNA accession no. gyrB accession no. S. oneidensis MR-1 ATCC 700550 TS Lake Oneida, NY, sediments AF005251 AF005694 S. oneidensis DLM7 Lower Green Bay sediments, Lake Michigan, Michigan AF387347 * AF005697 S. oneidenisis MR-4 Black Sea water column AF005252 AF005695 Shewanella sp. CL 256/73 NCTC 12093 Human cerebrospinal fluid AF387346 * AF378351 * S. putrefaciens str.

Techniques: Microarray, Hybridization