Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for <i>de Novo</i> DNA Synthesis

Recent advances in synthetic biology have resulted in an increasing demand for the <i>de novo</i> synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for <i>de novo</i> DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost <i>de novo</i> DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher 

Streamlining the Design-to-Build Transition with Build-Optimization Software Tools

Scaling-up capabilities for the design, build, and test of synthetic biology constructs holds great promise for the development of new applications in fuels, chemical production, or cellular-behavior engineering. Construct design is an essential component in this process; however, not every designed DNA sequence can be readily manufactured, even using state-of-the-art DNA synthesis methods. Current biological computer-aided design and manufacture tools (bioCAD/CAM) do not adequately consider the limitations of DNA synthesis technologies when generating their outputs. Designed sequences that violate DNA synthesis constraints may require substantial sequence redesign or lead to price-premiums and temporal delays, which adversely impact the efficiency of the DNA manufacturing process. We have developed a suite of build-optimization software tools (BOOST) to streamline the design-build transition in synthetic biology engineering workflows. BOOST incorporates knowledge of DNA synthesis success determinants into the design process to output ready-to-build sequences, preempting the need for sequence redesign. The BOOST web application is available at https://boost.jgi.doe.gov and its Application Program Interfaces (API) enable integration into automated, customized DNA design processes. The herein presented results highlight the effectiveness of BOOST in reducing DNA synthesis costs and timelines

Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP (<i>K</i>_D < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in <i>E. coli</i>. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems

Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP

Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (∼40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP (<i>K</i>_D < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in <i>E. coli</i>. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems

Structure of C₂₀ [5]-ladderane fatty acid, and the proposed major steps of the ladderane biosynthetic pathway.

<p>desaturation of acyl-ACPs to form polyunsaturated (all-<i>trans</i>) intermediates and cyclization <i>via</i> a radical cascade mechanism (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151087#pone.0151087.ref011" target="_blank">11</a>]).</p

Synthetic operons used in this study.

<p>Synthetic operons used in this study.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Growth and fatty acid profiles for strain expressing operons 1 and 2 and control strain.

<p>(A) Growth curve of ladd-initial and control strains. (B) GC/MS total ion chromatograms (TIC) of fatty acids extracted from ladd-initial and control strains post-cultivation and subjected to methyl ester derivatization. The most prominent fatty acid methyl esters are labeled with numbers: 1, C14:1; 2, C14:0; 3, C16:1; 4, C16:0; 5, C17 cyclopropane fatty acid (CFA); 6, C18:1; 7, C18:0; 8, C19 CFA.</p

DNA assembly scheme for construction of operons 3–11 (see Table 2 for additional detail).

<p>Each operon has a unique P_tet promoter, bicistronic design (BCD) element, and terminator chosen from the BIOFAB database. Restriction sites in each final operon plasmid allow for efficient, modular assembly of multiple operons in a final vector, such as a bacterial artificial chromosome or fosmid.</p

<i>In vivo</i> tests of function in putative phytoene desaturases from <i>K</i>. <i>stuttgartiensis</i> (kuste3336 and kuste3607).

<p>(Left) Phytoene desaturation to lycopene catalyzed by CrtI and schematic of the pLyc vector. (Right) Lycopene production in <i>E</i>. <i>coli</i> MG1655 strains (from left to right): Lyc (positive control), Lyc36 (<i>crtI</i> in pLyc replaced with kuste3336), Lyc07 (<i>crtI</i> in pLyc replaced with kuste3607), and Lyc-no-CrtI (negative control with <i>crtI</i> gene removed) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151087#pone.0151087.t001" target="_blank">Table 1</a> for details on strains).</p