3 research outputs found
Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies
Advances in synthetic biology and
genomics have enabled full-scale
genome engineering efforts on laboratory time scales. However, the
absence of sufficient approaches for mapping engineered genomes at
system-wide scales onto performance has limited the adoption of more
sophisticated algorithms for engineering complex biological systems.
Here we report on the development and application of a robust approach
to quantitatively map combinatorially engineered populations at scales
up to several dozen target sites. This approach works by assembling
genome engineered sites with cell-specific barcodes into a format
compatible with high-throughput sequencing technologies. This approach,
called barcoded-TRACE (bTRACE) was applied to assess <i>E. coli</i> populations engineered by recursive multiplex recombineering across
both 6-target sites and 31-target sites. The 31-target library was
then tracked throughout growth selections in the presence and absence
of isopentenol (a potential next-generation biofuel). We also use
the resolution of bTRACE to compare the influence of technical and
biological noise on genome engineering efforts
Codon Compression Algorithms for Saturation Mutagenesis
Saturation mutagenesis is employed
in protein engineering and genome-editing
efforts to generate libraries that span amino acid design space. Traditionally,
this is accomplished by using degenerate/compressed codons such as
NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop
codon. These solutions suffer from two types of redundancy: (a) different
codons for the same amino acid lead to bias, and (b) wild type amino
acid is included within the library. These redundancies increase library
size and downstream screening efforts. Here, we present a dynamic
approach to compress codons for any desired list of amino acids, taking
into account codon usage. This results in a unique codon collection
for every amino acid to be mutated, with the desired redundancy level.
Finally, we demonstrate that this approach can be used to design precise
oligo libraries amendable to recombineering and CRISPR-based genome
editing to obtain a diverse population with high efficiency
The Resistome: A Comprehensive Database of <i>Escherichia coli</i> Resistance Phenotypes
The
microbial ability to resist stressful environmental conditions
and chemical inhibitors is of great industrial and medical interest.
Much of the data related to mutation-based stress resistance, however,
is scattered through the academic literature, making it difficult
to apply systematic analyses to this wealth of information. To address
this issue, we introduce the Resistome database: a literature-curated
collection of <i>Escherichia coli</i> genotypes–phenotypes
containing over 5,000 mutants that resist hundreds of compounds and
environmental conditions. We use the Resistome to understand our current
state of knowledge regarding resistance and to detect potential synergy
or antagonism between resistance phenotypes. Our data set represents
one of the most comprehensive collections of genomic data related
to resistance currently available. Future development will focus on
the construction of a combined genomic–transcriptomic–proteomic
framework for understanding <i>E. coli</i>’s resistance
biology. The Resistome can be downloaded at https://bitbucket.org/jdwinkler/resistome_release/overview