Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function

Evaluation of Three Automated Genome Annotations for Halorhabdus utahensis

Genome annotations are accumulating rapidly and depend heavily on automated annotation systems. Many genome centers offer annotation systems but no one has compared their output in a systematic way to determine accuracy and inherent errors. Errors in the annotations are routinely deposited in databases such as NCBI and used to validate subsequent annotation errors. We submitted the genome sequence of halophilic archaeon Halorhabdus utahensis to be analyzed by three genome annotation services. We have examined the output from each service in a variety of ways in order to compare the methodology and effectiveness of the annotations, as well as to explore the genes, pathways, and physiology of the previously unannotated genome. The annotation services differ considerably in gene calls, features, and ease of use. We had to manually identify the origin of replication and the species-specific consensus ribosome-binding site. Additionally, we conducted laboratory experiments to test H. utahensis growth and enzyme activity. Current annotation practices need to improve in order to more accurately reflect a genome's biological potential. We make specific recommendations that could improve the quality of microbial annotation projects

Algorithms for automated DNA assembly

Generating a defined set of genetic constructs within a large combinatorial space provides a powerful method for engineering novel biological functions. However, the process of assembling more than a few specific DNA sequences can be costly, time consuming and error prone. Even if a correct theoretical construction scheme is developed manually, it is likely to be suboptimal by any number of cost metrics. Modular, robust and formal approaches are needed for exploring these vast design spaces. By automating the design of DNA fabrication schemes using computational algorithms, we can eliminate human error while reducing redundant operations, thus minimizing the time and cost required for conducting biological engineering experiments. Here, we provide algorithms that optimize the simultaneous assembly of a collection of related DNA sequences. We compare our algorithms to an exhaustive search on a small synthetic dataset and our results show that our algorithms can quickly find an optimal solution. Comparison with random search approaches on two real-world datasets show that our algorithms can also quickly find lower-cost solutions for large datasets

Scalable Plasmid Transfer using Engineered P1-based Phagemids

Dramatic improvements to computational, robotic, and biological tools have enabled genetic engineers to conduct increasingly sophisticated experiments. Further development of biological tools offers a route to bypass complex or expensive mechanical operations, thereby reducing the time and cost of highly parallelized experiments. Here, we engineer a system based on bacteriophage P1 to transfer DNA from one <i>E. coli</i> cell to another, bypassing the need for intermediate DNA isolation (e.g., minipreps). To initiate plasmid transfer, we refactored a native phage element into a DNA module capable of heterologously inducing phage lysis. After incorporating known <i>cis</i>-acting elements, we identified a novel <i>cis</i>-acting element that further improves transduction efficiency, exemplifying the ability of synthetic systems to offer insight into native ones. The system transfers DNAs up to 25 kilobases, the maximum assayed size, and operates well at microliter volumes, enabling manipulation of most routinely used DNAs. The system’s large DNA capacity and physical coupling of phage particles to phagemid DNA suggest applicability to biosynthetic pathway evolution, functional proteomics, and ultimately, diverse molecular biology operations including DNA fabrication

Glycolysis/gluconeogenesis pathway.

<p>Diagram based on RAST's KEGG pathway map displays present and absent enzymes for <i>H. utahensis</i>. Green boxes indicate that the enzyme was predicted by RAST and displayed in the KEGG map. Yellow boxes designate enzymes that were called by RAST but had not been added to the KEGG map. Red boxes mark enzymes that were listed as absent and could not be located in the <i>H. utahensis</i> genome.</p

Venn diagrams of gene predictions.

<p>(A) The diagram to the left shows the number of predicted protein coding genes that share stop sites with the other annotations. Overlapping regions indicate genes having same stop site between annotations. (B) The diagram to the right shows the number of predicted protein coding genes that share start site and stop site with the other annotations. Overlapping regions indicate genes having exact matches between annotations.</p

Comparison of rRNA calls.

<p>Review of predicted coding regions for ribosomal RNA for each annotation service shows that IMG and RAST have identical calls, while JCVI fails to call 23s rRNA and predicts different start and stop sites.</p

Potential comparison tool.

<p>Hypothetical comparison of start sites from multiple annotations, combined with species-specific RBS data. Start #2 would be the most likely start codon based on RBS spacing.</p

<i>H. utahensis</i> primary contig and ORC/Cdc6 genes.

<p>Circular display of the largest contig of the <i>H. utahensis</i> genome sequence. The contig begins at the top and wraps clockwise. The red bars illustrate the location of ORC/Cdc6 orthologs. The ORC/Cdc6 gene numbered 3 lies near the origin of replication, at 2,327,225 base pairs.</p