13 research outputs found
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Directed evolution of antimutator E. coli
Biological systems are essential tools for addressing societal challenges. There have been several successes in this field, however, a strong hindrance lies in the ephemeral nature of these systems ā cells are tiny factories that evolve. Evolution poses a problem because when a desired function is encoded into the DNA of the host organism, the host uses its own resources to perform the function and there is likely no associated fitness benefit. Therefore, there is strong selection for inactivation of the function due to the metabolic load imposed on cellular resources. One way to address this problem is to engineer evolutionary stability by lowering a host organismās basal mutation rate and concomitantly reducing the probability that an encoded function will become mutated.
In Chapter 1 of this dissertation, I discuss the nature of the metabolic cost associated with engineering biology and mechanisms by which host adaptation occurs. I also explore cellular pathways involved in genetic stability and examine previously characterized antimutators. Chapter 2 describes the first iteration of a directed evolution method used to engineer antimutators in Escherichia coli, Periodic Reselection for Evolutionarily Reliable Variants (PResERV). In this first PResERV experiment, I observe that the antimutator phenotype is due to mutations in genes involved DNA replication and RNA metabolism (polA, polB, and rne). In Chapter 3, I perform the same PResERV experiment on a greater scale and characterize a series of antimutator strains. The causative alleles in many of these strains are in genes involved in the tricarboxylic acid cycle and electron transport chain (sucD and sdhA). These alleles are shown to reduce oxidative stress. Chapter 4 demonstrates results from another PResERV experiment using a clean-genome E. coli strain, MDS42, as the host organism. In sum, this work shows the many mechanisms that lead to an antimutator phenotype, and these findings are used to build stable strains for reliable engineering of biology.
Finally, there are two appendices (Appendix A and B) which discuss my work in examining the evolutionary path to citrate utilization in Lenskiās long-term evolution experiment (LTEE) and a do-it-yourself method for using gellan gum as an alternative to microbial agar media.Microbiolog
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Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.
The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance
Bacterial Production of Gellan Gum as a Do-It-Yourself Alternative to Agar
Lack of access to reagents and equipment can make performing microbiology experiments difficult in K-12 classrooms and do-it-yourself (DIY) science settings. We demonstrate how the bacterium Sphingomonas paucimobilis can be used to synthesize gellan gum, an alternative to agar, with materials available to DIY scientists and educators. The method involves microwaving cultures of S. paucimobilis after a two-step growth procedure, supplementing them with additional media components as necessary, and then pouring plates. Gellan gum produced in the S. paucimobilis culture acts as a solidifying agent and provides a resilient surface that supports growth of microbes, including Escherichia coli and Saccharomyces cerevisiae. This procedure offers a unique opportunity to experiment with microbial production of an extracellular polysaccharide and to cheaply and sustainably source a reagent for research
Innovation in an <i>E</i>. <i>coli</i> evolution experiment is contingent on maintaining adaptive potential until competition subsides
<div><p>Key innovations are disruptive evolutionary events that enable a species to escape constraints and rapidly diversify. After 15 years of the Lenski long-term evolution experiment with <i>Escherichia coli</i>, cells in one of the twelve populations evolved the ability to utilize citrate, an abundant but previously untapped carbon source in the environment. Descendants of these cells became dominant in the population and subsequently diversified as a consequence of invading this vacant niche. Mutations responsible for the appearance of rudimentary citrate utilization and for refining this ability have been characterized. However, the complete nature of the genetic and/or ecological events that set the stage for this key innovation is unknown. In particular, it is unclear why it took so long for citrate utilization to evolve and why it still has evolved in only one of the twelve <i>E</i>. <i>coli</i> populations after 30 years of the Lenski experiment. In this study, we recapitulated the initial mutation needed to evolve citrate utilization in strains isolated from throughout the first 31,500 generations of the history of this population. We found that there was already a slight fitness benefit for this mutation in the original ancestor of the evolution experiment and in other early isolates. However, evolution of citrate utilization was blocked at this point due to competition with other mutations that improved fitness in the original niche. Subsequently, an anti-potentiated genetic background evolved in which it was deleterious to evolve rudimentary citrate utilization. Only later, after further mutations accumulated that restored the benefit of this first-step mutation and the overall rate of adaptation in the population slowed, was citrate utilization likely to evolve. Thus, intense competition and the types of mutations that it favors can lead to short-sighted evolutionary trajectories that hide a stepping stone needed to access a key innovation from many future generations.</p></div
Fitness consequences of evolving Cit<sup>+</sup> in different evolved genetic backgrounds.
<p><b>(a)</b> Results of the P<sub><i>rnk</i></sub><i>-citT</i> knock-in assay on 23 pre-Cit<sup>+</sup> evolved strains. The clones are ordered by the generation from which they were isolated. Error bars are 95% confidence intervals. Strain construction details and how the results of competition assays were combined into these fitness estimates are described in the <b>Methods</b> and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s001" target="_blank">S1</a>ā<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s004" target="_blank">S4</a> Figs</b>. <b>(b)</b> Increased lag phase upon addition of the P<sub><i>rnk</i></sub>-<i>citT</i> module in anti-potentiated strains. Growth curves for the ancestor, REL606, and two anti-potentiated strains, ZDB483 and ZDB14, are shown. Error bars are standard deviations of four replicate cultures.</p
Evolution of rudimentary citrate utilization by activating <i>citT</i> expression is slightly beneficial in the genetic background in which it evolved and in the LTEE ancestor.
<p><b>(a)</b> The <i>rnk</i>-<i>citG</i> duplication that evolved in the LTEE creates a genomic configuration in which a novel mRNA encoding the CitT transporter is expressed from the <i>rnk</i> promoter (P<i><sub>rnk</sub></i>) (right). This mutation alone is sufficient for weak citrate utilization (Cit<sup>+</sup> phenotype). It is the āactualizing mutationā in the evolution of this key innovation. Strain ZDB564 is the earliest Cit<sup>+</sup> isolate from the LTEE. In order to measure the effect that this mutation had on competitive fitness when it evolved, a spontaneous Cit<sup>ā</sup> revertant of ZDB564 in which the duplication collapsed back to the ancestral state was isolated (left). <b>(b)</b> Competitive fitness of Cit<sup>+</sup> versus Cit<sup>ā</sup> strain variants. The ZDB564 versus ZDB706 competitions measure the fitness effect of the <i>rnk</i>-<i>citG</i> duplication when it evolved. The ZDB706 and REL606 competitions test the effect of adding one copy of the evolved P<sub><i>rnk</i></sub>-<i>citT</i> module into a strain (+) versus adding an empty version of the same cassette (Ć), as pictured in <b>c</b>. An additional ZDB706 competition (in population) was conducted with the two strains together mixed at a 1:99 ratio with the evolved LTEE population from at 31,000 generations to determine if the mutation had a different effect on fitness when rare in the population. Starred strains (*) have a change to the Ara<sup>+</sup> marker state to allow competition with the corresponding Ara<sup>ā</sup> strain as illustrated in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s001" target="_blank">S1</a></b> and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s002" target="_blank">S2</a> Figs</b>. The marker change had no effect on competitive fitness in each case (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s003" target="_blank">S3 Fig</a>)</b>. Error bars are 95% confidence intervals. (<b>c</b>) Schematic of the gene cassettes used in the P<sub><i>rnk</i></sub>-<i>citT</i> knock-in assay showing how they were integrated into the <i>E</i>. <i>coli</i> chromosome in a way that replaces the native <i>lac</i> locus. <b>(d)</b> <i>citT</i> mRNA expression levels measured relative to the REL606 LTEE ancestor in the evolved Cit<sup>+</sup> isolate from the LTEE (ZDB564) and strains with the P<sub><i>rnk</i></sub>-<i>citT</i> and corresponding empty control cassettes integrated into their chromosomes. Error bars are 95% confidence intervals.</p
Potential for evolving Cit<sup>+</sup> mapped onto phylogeny.
<p>Phylogeny of isolates from the LTEE population including 20 new clones sequenced for this study to provide better resolution of the timing of mutations on the lineage leading to Cit<sup>+</sup> (names in italics). In order to identify changes in the degree of potentiation due to mutations, we mapped the results of the P<sub><i>rnk</i></sub><i>-citT</i> knock-in assay onto this phylogenetic tree. Colored symbols reflect the Cit<sup>+</sup> to Cit<sup>ā</sup> relative fitness measured for those strains. The ancestor and 61 evolved isolates were used to construct this phylogenetic tree (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s006" target="_blank">S1 Table</a></b>). Two clones isolated at 50,000 generations are not shown. Two strains that evolved citrate utilization in replay experiments under the LTEE conditions in a previous study [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.ref007" target="_blank">7</a>] are marked with plus signs (++), and three strains that had evolved alleles added or removed during strain construction as described in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007348#pgen.1007348.s007" target="_blank">S2 Table</a></b> are starred (*).</p
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Towards an informative mutant phenotype for every bacterial gene.
Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness
Towards an informative mutant phenotype for every bacterial gene.
Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness
Predicting the Genetic Stability of Engineered DNA Sequences with the EFM Calculator
Unwanted evolution can rapidly degrade
the performance of genetically
engineered circuits and metabolic pathways installed in living organisms.
We created the Evolutionary Failure Mode (EFM) Calculator to computationally
detect common sources of genetic instability in an input DNA sequence.
It predicts two types of mutational hotspots: deletions mediated by
homologous recombination and indels caused by replication slippage
on simple sequence repeats. We tested the performance of our algorithm
on genetic circuits that were previously redesigned for greater evolutionary
reliability and analyzed the stability of sequences in the iGEM Registry
of Standard Biological Parts. More than half of the parts in the Registry
are predicted to experience >100-fold elevated mutation rates due
to the inclusion of unstable sequence configurations. We anticipate
that the EFM Calculator will be a useful negative design tool for
avoiding volatile DNA encodings, thereby increasing the evolutionary
lifetimes of synthetic biology devices