31 research outputs found
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><sub>D</sub> < 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><sub>D</sub> < 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 C<sub>20</sub> [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
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<sub>tet</sub> 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