About
Activity
-
Also, check out Linna's paper (published in the same issue of Science) where one of the nanopore we designed was converted into a small-molecule…
Also, check out Linna's paper (published in the same issue of Science) where one of the nanopore we designed was converted into a small-molecule…
Liked by Marc Lajoie
-
In two new studies, we show that an effectively unlimited number of protein sensors can now be generated with the help of AI. This is a major step…
In two new studies, we show that an effectively unlimited number of protein sensors can now be generated with the help of AI. This is a major step…
Liked by Marc Lajoie
-
I'm happy to share that our nanopore design study is now published in Science! It took a lot of collaborative efforts from talented scientists in…
I'm happy to share that our nanopore design study is now published in Science! It took a lot of collaborative efforts from talented scientists in…
Liked by Marc Lajoie
Experience & Education
Publications
-
Genomically Recoded Organisms Expand Biological Functions
Science
We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating…
We describe the construction and characterization of a genomically recoded organism (GRO). We replaced all known UAG stop codons in Escherichia coli MG1655 with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids that expand the chemical diversity of proteins in vivo. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance.
Other authorsSee publication -
Probing the limits of genetic recoding in essential genes
Science
Engineering radically altered genetic codes will allow for genomically recoded organisms that have expanded chemical capabilities and are isolated from nature. We have previously reassigned the translation function of the UAG stop codon; however, reassigning sense codons poses a greater challenge because such codons are more prevalent, and their usage regulates gene expression in ways that are difficult to predict. To assess the feasibility of radically altering the genetic code, we selected a…
Engineering radically altered genetic codes will allow for genomically recoded organisms that have expanded chemical capabilities and are isolated from nature. We have previously reassigned the translation function of the UAG stop codon; however, reassigning sense codons poses a greater challenge because such codons are more prevalent, and their usage regulates gene expression in ways that are difficult to predict. To assess the feasibility of radically altering the genetic code, we selected a panel of 42 highly expressed essential genes for modification. Across 80 Escherichia coli strains, we removed all instances of 13 rare codons from these genes and attempted to shuffle all remaining codons. Our results suggest that the genome-wide removal of 13 codons is feasible; however, several genome design constraints were apparent, underscoring the importance of a strategy that rapidly prototypes and tests many designs in small pieces.
Other authorsSee publication -
Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria
Nucleic Acids Research
The discovery of diverse codon reassignment events has demonstrated that the canonical genetic
code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of…The discovery of diverse codon reassignment events has demonstrated that the canonical genetic
code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted tRNAAla UAG is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that tRNAAla UAG is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase
(AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNAAla; a G3A change in Ashbya
tRNAAla UAG abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae tRNAThr UAG confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.Other authorsSee publication -
Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases
PLOS One
Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease…
Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3′ ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.
Other authors -
-
Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering
Nucleic Acids Research
Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain…
Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.
Other authors -
-
Genome-wide Codon Replacement Using Synthetic Oligonucleotides and Engineered Conjugation
Science
We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed…
We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.
Other authors -
-
Tricyclic Compounds Containing Non-enolizable Cyano Enones. A Novel Class of Highly Potent Anti-inflammatory and Cytoprotective Agents
J. Med. Chem.
Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated…
Forty-four novel tricycles containing nonenolizable cyano enones (TCEs) were designed and synthesized on the basis of a semisynthetic pentacyclic triterpenoid, bardoxolone methyl, which is currently being developed in phase II clinical trials for the treatment of severe chronic kidney disease in diabetic patients. Most of the TCEs having two different kinds of nonenolizable cyano enones in rings A and C are highly potent suppressors of induction of inducible nitric oxide synthase stimulated with interferon-γ and are highly potent inducers of the cytoprotective enzymes heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Among these compounds, (±)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile ((±)-31) is the most potent in these bioassays in our pool of drug candidates including semisynthetic triterpenoids and synthetic tricycles. These facts strongly suggest that an essential factor for potency is not a triterpenoid skeleton but the cyano enone functionality. Notably, TCE 31 reduces hepatic tumorigenesis induced with aflatoxin in rats. Further preclinical studies and detailed mechanism studies on 31 are in progress.
Other authors -
-
Lambda Red Recombination in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate
GENETICS
The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5′ ends of…
The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5′ ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3′ overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.
Other authors -
Honors & Awards
-
35 Innovators Under 35
MIT Technology Review
-
Cancer Research Institute Irvington Postdoctoral Fellow
Cancer Research Institute
-
Washington Research Foundation Innovation Postdoctoral Fellow
Washington Research Foundation
-
Innovation and technology delegate
Academy of Achievement
-
30 under 30 in Science
Forbes Magazine
-
NDSEG fellowship
Department of Defense
More activity by Marc
-
I am excited to embark on this new journey! Thank you Sutro Biopharma, Inc., I feel privileged to be part of such an amazing team.
I am excited to embark on this new journey! Thank you Sutro Biopharma, Inc., I feel privileged to be part of such an amazing team.
Liked by Marc Lajoie
-
I am excited to announce that I have joined the External Innovation team at Lilly, where I will be focusing on our Immunology BD and Partnering…
I am excited to announce that I have joined the External Innovation team at Lilly, where I will be focusing on our Immunology BD and Partnering…
Liked by Marc Lajoie
-
Today we celebrate Associate Director P. Benjamin Stranges for an incredible 7 years at GRObio! Ben has been with the company since day 1 as our…
Today we celebrate Associate Director P. Benjamin Stranges for an incredible 7 years at GRObio! Ben has been with the company since day 1 as our…
Liked by Marc Lajoie
-
In recognition of Alex Badamchi-Zadeh, PhD's contribution to EyeBio’s success via his role as a core member of the SV deal team and an observer on…
In recognition of Alex Badamchi-Zadeh, PhD's contribution to EyeBio’s success via his role as a core member of the SV deal team and an observer on…
Liked by Marc Lajoie
-
Recognizing our brilliant Johnson & Johnson Innovative Medicine employees for their outstanding achievements in science, innovation, and inclusive…
Recognizing our brilliant Johnson & Johnson Innovative Medicine employees for their outstanding achievements in science, innovation, and inclusive…
Liked by Marc Lajoie
-
I'm proud to announce that our portfolio company EyeBio has been acquired by Merck for $1.3B upfront and up to $3B, just 7 months after our…
I'm proud to announce that our portfolio company EyeBio has been acquired by Merck for $1.3B upfront and up to $3B, just 7 months after our…
Liked by Marc Lajoie
-
The cat is out of the bag!! Amplitude Ventures has closed on Fund II, bringing our total AUM to >$500M across 2 funds, less than 5 years from our…
The cat is out of the bag!! Amplitude Ventures has closed on Fund II, bringing our total AUM to >$500M across 2 funds, less than 5 years from our…
Liked by Marc Lajoie
-
So proud of our DSP and ML Research Scientist who presented her poster at a conference this week. And way to drive home the Wavely spirit by wearing…
So proud of our DSP and ML Research Scientist who presented her poster at a conference this week. And way to drive home the Wavely spirit by wearing…
Liked by Marc Lajoie
-
Very excited to announce the expansion of our partnership with Lawrence Livermore National Laboratory with an additional $14.5M from the United…
Very excited to announce the expansion of our partnership with Lawrence Livermore National Laboratory with an additional $14.5M from the United…
Liked by Marc Lajoie
-
This is amazing. Three of our terrific FogPharma employees gained admission to top graduate schools this year! Wishing Gregory Valtierra great…
This is amazing. Three of our terrific FogPharma employees gained admission to top graduate schools this year! Wishing Gregory Valtierra great…
Liked by Marc Lajoie
-
it is important for policy-makers and other government stakeholders to gain a more comprehensive understanding of the biotech ecosystem in order to…
it is important for policy-makers and other government stakeholders to gain a more comprehensive understanding of the biotech ecosystem in order to…
Liked by Marc Lajoie
-
Today is my last day of an incredible journey. I want to express my gratitude to the Celgene Pharma/Bristol Myers Squibb family for a great 9 year…
Today is my last day of an incredible journey. I want to express my gratitude to the Celgene Pharma/Bristol Myers Squibb family for a great 9 year…
Liked by Marc Lajoie
Other similar profiles
Explore collaborative articles
We’re unlocking community knowledge in a new way. Experts add insights directly into each article, started with the help of AI.
Explore MoreOthers named Marc Lajoie in United States
-
Marc Lajoie
Data journalist
-
Marc Lajoie
Regional Project Leader
-
Marc Lajoie
Manager of Procurement at Imperial Distributors
-
Marc LaJoie
Owner, The arlington
10 others named Marc Lajoie in United States are on LinkedIn
See others named Marc Lajoie