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 function. 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.

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 , 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.

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.

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.

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.

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.

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.

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.

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.

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.