Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function1-3. To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis1-from five episomes in 10 days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly5,6, along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections1,7,8, we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months.

Sense codon reassignment enables viral resistance and encoded polymer synthesis.

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS.

We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7-10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR-Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.

Total synthesis of Escherichia coli with a recoded genome.

Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA.

Axon-Dependent Patterning and Maintenance of Somatosensory Dendritic Arbors.

The mechanisms that pattern and maintain dendritic arbors are key to understanding the principles that govern nervous system assembly. The activity of presynaptic axons has long been known to shape dendrites, but activity-independent functions of axons in this process have remained elusive. Here, we show that in Caenorhabditis elegans, the axons of the ALA neuron control guidance and extension of the 1° dendrites of PVD somatosensory neurons independently of ALA activity. PVD 1° dendrites mimic ALA axon guidance defects in loss-of-function mutants for the extracellular matrix molecule MIG-6/Papilin or the UNC-6/Netrin pathway, suggesting that axon-dendrite adhesion is important for dendrite formation. We found that the SAX-7/L1CAM cell adhesion molecule engages in distinct molecular mechanisms to mediate extensions of PVD 1° dendrites and maintain the ALA-PVD axon-dendritic fascicle, respectively. Thus, axons can serve as critical scaffolds to pattern and maintain dendrites through contact-dependent but activity-independent mechanisms.

Defining synonymous codon compression schemes by genome recoding.

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions, and the absence of methods that enable the stepwise replacement of the Escherichia coli genome with long synthetic DNA and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress towards this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (>100-kb) synthetic DNA, through the in vivo excision of double-stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda-red-mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.

Identification of novel protein functions and signaling mechanisms by genetics and quantitative phosphoproteomics in Caenorhabditis elegans.

Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment.

A method for measuring fatty acid oxidation in C. elegans.

The nematode C. elegans has during the past decade proven to be a valuable model organism to identify and examine molecular mechanisms regulating lipid storage and metabolism. While the primary approach has been to identify genes and pathways conferring alterations in lipid accumulation, only a few recent studies have recognized the central role of fatty acid degradation in cellular lipid homeostasis. In the present study, we show how complete oxidation of fatty acids can be determined in live C. elegans by examining oxidation of tritium-labeled fatty acids to tritiated H2O that can be measured by scintillation counting. Treating animals with sodium azide, an inhibitor of the electron transport chain, reduced (3)H2O production to approximately 15%, while boiling of animals prior to assay completely blocked the production of labeled water. We demonstrate that worms fed different bacterial strains exhibit different fatty acid oxidation rates. We show that starvation results in increased fatty acid oxidation, which is independent of the transcription factor NHR-49. On the contrary, fatty acid oxidation is reduced to approximately 70% in animals lacking the worm homolog of the insulin receptor, DAF-2. Hence, the present methodology can be used to delineate the role of specific genes and pathways in the regulation of ß-oxidation in C. elegans.

Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans.

Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology to quantitatively examine metabolic and signaling pathways in yeast, fruit flies, plants, cell cultures and mice. However, only metabolic labeling using (15)N has been applied to examine such events in the nematode Caenorhabditis elegans. We have recently shown that C. elegans can be completely labeled with heavy-labeled lysine by feeding worms on prelabeled lysine auxotroph Escherichia coli for just one generation. We applied this methodology to examine the organismal response to functional loss or RNAi mediated knock down of the transcription factor NHR-49, and found numerous proteins involved in lipid metabolism to be downregulated, which is consistent with its previously proposed function as a transcriptional regulator of fatty acid metabolism. The combined use of quantitative proteomics and selective gene knockdown by RNAi provides a powerful tool with broad implications for C. elegans biology.

Quantitative proteomics by amino acid labeling in C. elegans.

We demonstrate labeling of Caenorhabditis elegans with heavy isotope-labeled lysine by feeding them with heavy isotope-labeled Escherichia coli. Using heavy isotope-labeled worms and quantitative proteomics methods, we identified several proteins that are regulated in response to loss or RNAi-mediated knockdown of the nuclear hormone receptor 49 in C. elegans. The combined use of quantitative proteomics and selective gene knockdown is a powerful tool for C. elegans biology.