Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871 bp designer eukaryotic chromosome, synIII, which is based on the 316,617 bp native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, tRNAs, transposons and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in “a mater” derivatives resulting from loss of the MATα allele on synIII. The total synthesis of synIII represents the first complete design and synthesis of a eukaryotic chromosome, establishing S. cerevisiae as the basis for designer eukaryotic genome biology.

Proteomic analysis can be a critical bottleneck in cellular characterization. The current paradigm relies primarily on mass spectrometry of peptides and affinity reagents (i.e. antibodies), both of which require a priori knowledge of the sample. A non-biased protein sequencing method, with a dynamic range that covered the full range of protein concentrations in proteomes, would revolutionize the field of proteomics, allowing a more facile characterization of novel gene products and subcellular complexes. To this end, several new platforms based on single-molecule protein-sequencing approaches have been proposed. This review summarizes four of these approaches, highlighting advantages, limitations and challenges for each method towards advancing as a core technology for next-generation protein sequencing.

The switch-like regulation of protein activity by molecular signals is abundant in native proteins. The ability to engineer proteins with novel regulation has applications in bio-sensors, selective protein therapeutics, and basic research. One approach to building proteins with novel switch properties is creating combinatorial libraries of gene fusions between genes encoding proteins that have the prerequisite input and output functions of the desired switch. These libraries are then subjected to selections and/or screens to identify those rare gene fusions that encode functional switches. Combinatorial libraries in which an insert gene is inserted randomly into an acceptor gene have been useful for creating switches, particularly when combined with circular permutation of the insert gene. Methods for creating random domain insertion libraries are described. Three methods for creating a diverse set of insertion sites in the acceptor gene are presented and compared: DNase I digestion, S1 nuclease digestion, and multiplex inverse PCR. A PCR-based method for creating a library of circular permutations of the insert gene is also presented.

Protein switches have a variety of potential applications in biotechnology and medicine that motivate efforts to accelerate their development. Switches can be built by the proper fusion of two proteins with the prerequisite input and output functions. However, the exact fusion geometry for switch creation, which typically involves insertion of one protein domain into the other, is difficult to predict. Based on our previous work developing protein switches using periplasmic binding proteins as input domains, we wondered whether there are "hot spots" for insertion of output domains and successful switch creation within this class of proteins. Here we describe directed evolution experiments that identified switches in which TEM-1 beta-lactamase (BLA) is inserted into the class I periplasmic binding proteins ribose binding protein (RBP), glucose binding protein (GBP), and xylose binding protein (XBP). Although some overlap in sites for successful switch insertion could be found among the paralogs, successful switches at these sites required different linkers between the domains and different circular permutations of the BLA protein. Our data suggests that sites for successful switch creation are not easily transferable between paralogs. Furthermore, by comparison to a previous study in which switches were created by inserting a xylanase into XBP, we find no correlation between insertion sites when using two different output domains. We conclude that the switch property likely depends on the precise molecular details of the fusions and cannot be easily predicted from some overall general structural property of the fusion topology.