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    New England Biolabs e2611s
    A large-scale DNA data storage in living cell. a) The workflow for the manufacture of <t>mixed</t> culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to <t>assembly</t> and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original <t>master</t> pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.
    E2611s, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1334 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A large-scale DNA data storage in living cell. a) The workflow for the manufacture of mixed culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to assembly and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original master pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.

    Journal: bioRxiv

    Article Title: Mixed Culture of Bacterial Cell for Large Scale DNA Storage

    doi: 10.1101/2020.02.21.960476

    Figure Lengend Snippet: A large-scale DNA data storage in living cell. a) The workflow for the manufacture of mixed culture living cell data storage materials. Oligo pool was assembled with 1E+6⁓7 of average copy of each oligo was subjected to assembly and then transformed into E. coli cell with about 1E+1⁓2 average colony number of each oligo was obtained and then the cell population could be amplified to large scale in mixed culture for further plasmid retrieve and information decoding. b) the 0.9% dropout oligos in 1 st passaging of one fragment assembly (red line) and the 0.56% dropout oligos in 10x sequencing reads of original master pool (blue line) were mapped to the oligo frequency distribution of original master pool (gray line). c) In comparison with previous reported major systems for DNA storage in living cell including 0.25 kbps by Yachie in 2007, 18.2 bps by Shipman in 2017 and 2.8 kbps by Sun in 2019, totally 97.7 kbps DNA for 509 oligos pool and 2304 kbps for 11520 oligos pool were stored in mixed culture of E. coli cells at cost lower than 1E-4$ per base and mixed cell storage materials could be manufactured within 24 hrs.

    Article Snippet: Then the Gibson Assembly® Master Mix – Assembly (NEB, #E2611) was used according to user’s manual.

    Techniques: Transformation Assay, Amplification, Plasmid Preparation, Passaging, Sequencing

    CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

    Journal: EvoDevo

    Article Title: CRISPR-based gene replacement reveals evolutionarily conserved axon guidance functions of Drosophila Robo3 and Tribolium Robo2/3

    doi: 10.1186/s13227-017-0073-y

    Figure Lengend Snippet: CRISPR-based gene replacement of robo3. a Schematic of the robo3 gene showing intron/exon structure and location of gRNA target sites, robo3 TcRobo2/3 homologous donor plasmid, and the final modified robo3 TcRobo2/3 allele. Endogenous robo3 coding exons are shown as purple boxes ; 5′ and 3′ untranslated regions are shown as light gray boxes . The start of transcription is indicated by the bent arrow . Introns and exons are shown to scale, with the exception of the first intron, from which approximately 13 kb has been omitted. Red arrows indicate the location of upstream (gRNA 1) and downstream (gRNA 2) gRNA target sites. Gray brackets demarcate the region to be replaced by sequences from the donor plasmid. Arrows indicate the position and orientation of PCR primers. b Partial DNA sequences of the unmodified robo3 gene and the modified robo3 TcRobo2/3 allele. Black letters indicated endogenous DNA sequence; red letters indicate exogenous sequence. Both DNA strands are illustrated. The gRNA protospacer and PAM sequences are indicated for both gRNAs. The first five base pairs of robo3 exon 2 are unaltered in the robo3 TcRobo2/3 allele, and the robo3 coding sequence beginning with codon H21 is replaced by the HA-tagged TcRobo2/3 cDNA. The endogenous robo3 transcription start site, ATG start codon, and signal peptide are retained in exon 1. The PAM sequences and portions of both protospacers are deleted in the modified allele, ensuring that the robo3 TcRobo2/3 donor plasmid and modified robo3 TcRobo2/3 allele are not targeted by Cas9. UTR untranslated regions, 5 ′ H 5′ homology region, 3′H 3′ homology region, HA hemagglutinin epitope tag, gRNA guide RNA, HDR homology-directed repair, PAM protospacer adjacent motif

    Article Snippet: Construction of robo3TcRobo2/3 donor plasmid The initial robo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611).

    Techniques: CRISPR, Plasmid Preparation, Modification, Polymerase Chain Reaction, Sequencing