Millstone: software for multiplex microbial genome analysis and engineering

Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.; Ahern, Brian W.; Napolitano, Michael G.; Chen, Kevin Y.; Chen, Changping; Church, George M.

doi:10.1186/s13059-017-1223-1

Cited by 5 publications

(2 citation statements)

References 18 publications

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“…Unusually high coverage results from incorrect mapping of short reads to high-copy sequences such as insertion elements and ribosomal RNA. To determine the abundance of retron donors conferring SNPs, reads from amplicons obtained after selection were aligned to the MG1655 reference genome, and SNPs were inferred using Millstone (65). Depth of SNP coverage in the resulting output reflects abundance in the pool, and was used for visualization and analysis.…”

Section: Methodsmentioning

confidence: 99%

High-throughput functional variant screens via in vivo production of single-stranded DNA

Schubert

Goodman

Wannier

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

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Section: Methodsmentioning

confidence: 99%

High-throughput functional variant screens via in vivo production of single-stranded DNA

Schubert

Goodman

Wannier

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…CP006698.1) and the sequence of the plasmid encoding nsAA incorporation machinery. The Millstone software suite was used to identify variants, provide measures of sequencing confidence, and predict their likelihood of altering gene function ( 49 ). Genomic variants of low confidence, low sequence coverage, or presence in the ancestral strain were discarded, prioritizing variants observed in three nonessential genes that encode membrane proteins: acrB , emrD , and trkH .…”

Section: Methodsmentioning

confidence: 99%

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

et al. 2021

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Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

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Long-term adaptive evolution of genomically recodedEscherichia coli

et al. 2017

Preprint

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Efforts are underway to construct several recoded genomes anticipated to exhibit multi-virus resistance, enhanced non-standard amino acid (NSAA) incorporation, and capability for synthetic biocontainment. Though we succeeded in pioneering the first genomically recoded organism (Escherichia coli strain C321.∆A), its fitness is far lower than that of its non-recoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for NSAA-linked applications requiring defined media such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceed the growth rates of both the ancestral C321.∆A and non-recoded strains, permitting use of the recoded chassis in several new contexts. We use next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstruct individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occur only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared to application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/162834 doi: bioRxiv preprint first posted online Jul. 12, 2017; Significance Statement:After demonstrating construction of an organism with an altered genetic code, we sought to evolve this organism for many generations to improve its fitness and learn what unique changes natural selection would bestow upon it. Although this organism initially had impaired fitness, we observed that adaptive laboratory evolution resulted in several selective mutations that corrected for insufficient translation termination and for unintended mutations that occurred when originally altering the genetic code. This work further bolsters our understanding of the pliability of the genetic code, it will help guide ongoing and future efforts seeking to recode genomes, and it results in a useful strain for non-standard amino acid incorporation in numerous contexts relevant for research and industry.peer-reviewed)

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Millstone: software for multiplex microbial genome analysis and engineering

Cited by 5 publications

References 18 publications

High-throughput functional variant screens via in vivo production of single-stranded DNA

High-throughput functional variant screens via in vivo production of single-stranded DNA

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells

Long-term adaptive evolution of genomically recodedEscherichia coli

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