A nucleoside kinase as a dual selector for genetic switches and circuits

The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30,000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits.     

An efficient platform for genetic selection and screening of gene switches in Escherichia coli

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.     

Development of a genetic selection for catalytic antibodies

The design and evaluation of a new genetic selection system for evolving catalytic antibodies with aldolase activity are described. Through a series of model selections, we have identified selection conditions where expression of a catalytically active antibody confers a growth advantage to Escherichia coli. In addition, we provide evidence that the growth advantage is a direct result of catalytic activity.     

Methods to create a stringent selection system for mammalian cell lines

The efficient establishment of high protein producing recombinant mammalian cell lines is facilitated by the use of a stringent selection system. Here, we describe two methods to create a stringent selection system based on the Zeocin resistance marker. First, we cloned increasingly longer stretches of DNA, encoding a range of 8-131 amino acids immediately upstream of the Zeocin selection marker gene. The DNA stretches were separated from the open reading frame of the selection marker gene by a stopcodon. The idea behind this was that the translation machinery will first translate the small peptide, stop and then restart at the AUG of the Zeocin marker. This process, however, will become less efficient with increasingly longer stretches of DNA upstream of the Zeocin marker that has to be translated first. This would result in lower levels of the Zeocin selection marker protein and thus a higher selection stringency of the system. Secondly, we performed a genetic screen to identify PCR induced mutations in the Zeocin selection protein that functionally impair the selection marker protein. Both the insertion of increasingly longer peptides and several Zeocin selection protein mutants resulted in a decreasing number of stably transfected colonies that concomitantly displayed higher protein expression levels. When the Zeocin mutants were combined with very short small peptides (8-14 amino acids long), this created a flexible, high stringency selection system. The system allows the rapid establishment of few, but high protein producing mammalian cell lines.     

Incomplete loss of a conserved trait: function,latitudinal cline,and genetic constraints

Retention of nonfunctional traits over evolutionary time is puzzling, because the cost of trait production should drive loss. Indeed, several studies have found nonfunctional traits are rapidly eliminated by selection. However, theory suggests that complex genetic interactions and a lack of genetic variance can constrain evolution, including trait loss. In the mustard family Brassicaceae the conserved floral condition includes four long and two short stamens, but we show that short stamens in the highly self‐pollinating mustard Arabidopsis thaliana do not significantly increase selfed seed set, suggesting that the trait has lost most or all of its function after the transition to selfing. We find that short stamen loss is common in native populations. Loss is incomplete and decreases with increasing latitude, a cline unexplained by correlations with flowering time or ovule count (which also vary with latitude). Using recombinant inbred lines derived from a cross between plants at the latitudinal extremes of the native range, we found three QTLs affecting short stamen number, with epistasis among them constraining stamen loss. Constraints on stamen loss from both epistasis and low genetic variance may be augmented by high selfing rates, suggesting that these kinds of constraints may be common in inbred species.     

Rapid and Liquid-Based Selection of Genetic Switches Using Nucleoside Kinase Fused with Aminoglycoside Phosphotransferase

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3’)-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.     

The interpretation of selection coefficients

Evolutionary biologists have an array of powerful theoretical techniques that can accurately predict changes in the genetic composition of populations. Changes in gene frequencies and genetic associations between loci can be tracked as they respond to a wide variety of evolutionary forces. However, it is often less clear how to decompose these various forces into components that accurately reflect the underlying biology. Here, we present several issues that arise in the definition and interpretation of selection and selection coefficients, focusing on insights gained through the examination of selection coefficients in multilocus notation. Using this notation, we discuss how its flexibility—which allows different biological units to be identified as targets of selection—is reflected in the interpretation of the coefficients that the notation generates. In many situations, it can be difficult to agree on whether loci can be considered to be under “direct” versus “indirect” selection, or to quantify this selection. We present arguments for what the terms direct and indirect selection might best encompass, considering a range of issues, from viability and sexual selection to kin selection. We show how multilocus notation can discriminate between direct and indirect selection, and describe when it can do so.     

Recent selection for self‐compatibility in a population of Leavenworthia alabamica

The evolution of self‐compatibility (SC) is the first step in the evolutionary transition in plants from outcrossing enforced by self‐incompatibility (SI) to self‐fertilization. In the Brassicaceae, SI is controlled by alleles of two tightly linked genes at the S‐locus. Despite permitting inbreeding, mutations at the S‐locus leading to SC may be selected if they provide reproductive assurance and/or gain a transmission advantage in a population when SC plants self‐ and outcross. Positive selection can leave a genomic signature in the regions physically linked to the focus of selection when selection has occurred recently. From an SC population of Leavenworthia alabamica with a known nonfunctional mutation at the S‐locus, we collected sequence data from a ～690 Kb region surrounding the S‐locus, as well as from regions not linked to the S‐locus. To test for recent positive selection acting at the S‐locus, we examined polymorphism and the site‐frequency spectra. Using forward simulations, we demonstrate that recent selection of the strength expected for SC at a locus formerly under balancing selection can generate patterns similar to those seen in our empirical data.     

A plasmid display platform for the selection of peptides exhibiting a functional cell-penetrating phenotype

Cell-penetrating peptides (CPPs) represent a promising nonviral platform for the delivery of therapeutic cargos to cells and tissues. However, these peptides are often nonspecific, and their mechanism of action is still a subject of debate, which hinders the design of new CPPs. The alternative to rational protein design is the combinatorial approach to protein engineering, whereby large libraries of peptides are created and a screening or selection procedure is used to identify members with the desired phenotype(s). Here we describe a novel procedure for selecting peptides with a CPP phenotype using a plasmid display (PD) platform to link the peptides to their encoding DNA sequences. The PD system is based on genetic fusions to a DNA binding domain. The plasmid was designed to concomitantly express a fluorescent reporter protein to serve as a mock therapeutic cargo indicating its functional delivery into a cell. We have demonstrated this selection strategy using a control CPP (the TAT peptide) in the PC12 neuronal-like cell line. In the absence of transfection reagents, TAT was unable to deliver the protein/DNA complexes. The inclusion of the HA2 peptide from the hemagglutinin protein and the addition of polyethylenimine (PEI) were similarly ineffective. The addition of Lipofectamine, however, enabled the TAT-mediated delivery of the protein/DNA complexes, which was significantly better than control experiments without a CPP. This new PD selection platform will be a valuable new approach for use in identifying unique CPPs from randomized libraries with novel abilities and specificities.     

Beyond directed evolution: Darwinian selection as a tool for synthetic biology

Synthetic biology is an engineering approach that seeks to design and construct new biological parts, devices and systems, as well as to re-design existing components. However, rationally designed synthetic circuits may not work as expected due to the context-dependence of biological parts. Darwinian selection, the main mechanism through which evolution works, is a major force in creating biodiversity and may be a powerful tool for synthetic biology. This article reviews selection-based techniques and proposes strict Darwinian selection as an alternative approach for the identification and characterization of parts. Additionally, a strategy for fine-tuning of relatively complex circuits by coupling them to a master standard circuit is discussed.     

An analytical contrast between fitness maximization and selection for mixability

It has been recently shown numerically that sex enables selection for alleles that perform well across different genetic contexts, i.e., selection for mixability. Here we capture this result analytically in a simple case. This serves a dual purpose. First, it provides a clear distinction between fitness maximization and selection for mixability. Second, it points out a limitation of the traditional analytical approach as applied to mixability.     

A map of recent positive selection in the human genome

The identification of signals of very recent positive selection provides information about the adaptation of modern humans to local conditions. We report here on a genome-wide scan for signals of very recent positive selection in favor of variants that have not yet reached fixation. We describe a new analytical method for scanning single nucleotide polymorphism (SNP) data for signals of recent selection, and apply this to data from the International HapMap Project. In all three continental groups we find widespread signals of recent positive selection. Most signals are region-specific, though a significant excess are shared across groups. Contrary to some earlier low resolution studies that suggested a paucity of recent selection in sub-Saharan Africans, we find that by some measures our strongest signals of selection are from the Yoruba population. Finally, since these signals indicate the existence of genetic variants that have substantially different fitnesses, they must indicate loci that are the source of significant phenotypic variation. Though the relevant phenotypes are generally not known, such loci should be of particular interest in mapping studies of complex traits. For this purpose we have developed a set of SNPs that can be used to tag the strongest ∼250 signals of recent selection in each population.     

Testing for a genetic response to sexual selection in a wild Drosophila population

In accordance with the consensus that sexual selection is responsible for the rapid evolution of display traits on macroevolutionary scales, microevolutionary studies suggest sexual selection is a widespread and often strong form of directional selection in nature. However, empirical evidence for the contemporary evolution of sexually selected traits via sexual rather than natural selection remains weak. In this study, we used a novel application of quantitative genetic breeding designs to test for a genetic response to sexual selection on eight chemical display traits from a field population of the fly, Drosophila serrata. Using our quantitative genetic approach, we were able to detect a genetically based difference in means between groups of males descended from fathers who had either successfully sired offspring or were randomly collected from the same wild population for one of these display traits, the diene (Z,Z)‐5,9‐C_27 : 2. Our experimental results, in combination with previous laboratory studies on this system, suggest that both natural and sexual selection may be influencing the evolutionary trajectories of these traits in nature, limiting the capacity for a contemporary evolutionary response.     

Multilevel selection 2: Estimating the genetic parameters determining inheritance and response to selection

Interactions among individuals are universal, both in animals and in plants and in natural as well as domestic populations. Understanding the consequences of these interactions for the evolution of populations by either natural or artificial selection requires knowledge of the heritable components underlying them. Here we present statistical methodology to estimate the genetic parameters determining response to multilevel selection of traits affected by interactions among individuals in general populations. We apply these methods to obtain estimates of genetic parameters for survival days in a population of layer chickens with high mortality due to pecking behavior. We find that heritable variation is threefold greater than that obtained from classical analyses, meaning that two-thirds of the full heritable variation is hidden to classical analysis due to social interactions. As a consequence, predicted responses to multilevel selection applied to this population are threefold greater than classical predictions. This work, combined with the quantitative genetic theory for response to multilevel selection presented in an accompanying article in this issue, enables the design of selection programs to effectively reduce competitive interactions in livestock and plants and the prediction of the effects of social interactions on evolution in natural populations undergoing multilevel selection.     

A coalescent dual process in a Moran model with genic selection

A coalescent dual process for a multi-type Moran model with genic selection is derived using a generator approach. This leads to an expansion of the transition functions in the Moran model and the Wright–Fisher diffusion process limit in terms of the transition functions for the coalescent dual. A graphical representation of the Moran model (in the spirit of Harris) identifies the dual as a strong dual process following typed lines backwards in time. An application is made to the harmonic measure problem of finding the joint probability distribution of the time to the first loss of an allele from the population and the distribution of the surviving alleles at the time of loss. Our dual process mirrors the Ancestral Selection Graph of [Krone, S. M., Neuhauser, C., 1997. Ancestral processes with selection. Theoret. Popul. Biol. 51, 210–237; Neuhauser, C., Krone, S. M., 1997. The genealogy of samples in models with selection. Genetics 145, 519–534], which allows one to reconstruct the genealogy of a random sample from a population subject to genic selection. In our setting, we follow [Stephens, M., Donnelly, P., 2002. Ancestral inference in population genetics models with selection. Aust. N. Z. J. Stat. 45, 395–430] in assuming that the types of individuals in the sample are known. There are also close links to [Fearnhead, P., 2002. The common ancestor at a nonneutral locus. J. Appl. Probab. 39, 38–54]. However, our methods and applications are quite different. This work can also be thought of as extending a dual process construction in a Wright–Fisher diffusion in [Barbour, A.D., Ethier, S.N., Griffiths, R.C., 2000. A transition function expansion for a diffusion model with selection. Ann. Appl. Probab. 10, 123–162]. The application to the harmonic measure problem extends a construction provided in the setting of a neutral diffusion process model in [Ethier, S.N., Griffiths, R.C., 1991. Harmonic measure for random genetic drift. In: Pinsky, M.A. (Ed.), Diffusion Processes and Related Problems in Analysis, vol. 1. In: Progress in Probability Series, vol. 22, Birkhäuser, Boston, pp. 73–81].     

Variation,selection and evolution of function-valued traits

We describe an emerging framework for understanding variation, selection and evolution of phenotypic traits that are mathematical functions. We use one specific empirical example – thermal performance curves (TPCs) for growth rates of caterpillars – to demonstrate how models for function-valued traits are natural extensions of more familiar, multivariate models for correlated, quantitative traits. We emphasize three main points. First, because function-valued traits are continuous functions, there are important constraints on their patterns of variation that are not captured by multivariate models. Phenotypic and genetic variation in function-valued traits can be quantified in terms of variance-covariance functions and their associated eigenfunctions: we illustrate how these are estimated as well as their biological interpretations for TPCs. Second, selection on a function-valued trait is itself a function, defined in terms of selection gradient functions. For TPCs, the selection gradient describes how the relationship between an organism's performance and its fitness varies as a function of its temperature. We show how the form of the selection gradient function for TPCs relates to the frequency distribution of environmental states (caterpillar temperatures) during selection. Third, we can predict evolutionary responses of function-valued traits in terms of the genetic variance-covariance and the selection gradient functions. We illustrate how non-linear evolutionary responses of TPCs may occur even when the mean phenotype and the selection gradient are themselves linear functions of temperature. Finally, we discuss some of the methodological and empirical challenges for future studies of the evolution of function-valued traits.     

Environmentally induced changes in correlated responses to selection reveal variable pleiotropy across a complex genetic network

Selection in novel environments can lead to a coordinated evolutionary response across a suite of characters. Environmental conditions can also potentially induce changes in the genetic architecture of complex traits, which in turn could alter the pattern of the multivariate response to selection. We describe a factorial selection experiment using the nematode Caenorhabditis remanei in which two different stress‐related phenotypes (heat and oxidative stress resistance) were selected under three different environmental conditions. The pattern of covariation in the evolutionary response between phenotypes or across environments differed depending on the environment in which selection occurred, including asymmetrical responses to selection in some cases. These results indicate that variation in pleiotropy across the stress response network is highly sensitive to the external environment. Our findings highlight the complexity of the interaction between genes and environment that influences the ability of organisms to acclimate to novel environments. They also make clear the need to identify the underlying genetic basis of genetic correlations in order understand how patterns of pleiotropy are distributed across complex genetic networks.     

The depletion of genetic variance by sexual selection

Sexually selected traits display substantial genetic variance [1, 2], in conflict with the expectation that sexual selection will deplete it [3-5]. Condition dependence is thought to resolve this paradox [5-7], but experimental tests that relate the direction of sexual selection to the availability of genetic variance are lacking. Here, we show that condition-dependent expression is not sufficient to maintain genetic variance available to sexual selection in multiple male sexually selected traits. We employed an experimental design that simultaneously determined the quantitative genetic basis of nine male cuticular hydrocarbons (CHCs) of Drosophila bunnanda, the extent of condition dependence of these traits, and the strength and direction of sexual selection acting upon them. The CHCs of D. bunnanda are condition dependent, with 18% of the genetic variance in male body size explained by genetic variance in CHCs. Despite the presence of genetic variance in individual male traits, 98% of the genetic variance in CHCs was found to be orientated more than 88 degrees away from the direction of sexual selection and therefore unavailable to selection. A lack of genetic variance in male traits in the direction of sexual selection may represent a general feature of sexually selected systems, even in the presence of condition-dependent trait expression.     

A coalescent dual process in a Moran model with genic selection, and the lambda coalescent limit

The genealogical structure of neutral populations in which reproductive success is highly-skewed has been the subject of many recent studies. Here we derive a coalescent dual process for a related class of continuous-time Moran models with viability selection. In these models, individuals can give birth to multiple offspring whose survival depends on both the parental genotype and the brood size. This extends the dual process construction for a multi-type Moran model with genic selection described in Etheridge and Griffiths (2009). We show that in the limit of infinite population size the non-neutral Moran models converge to a Markov jump process which we call the Λ-Fleming-Viot process with viability selection and we derive a coalescent dual for this process directly from the generator and as a limit from the Moran models. The dual is a branching-coalescing process similar to the Ancestral Selection Graph which follows the typed ancestry of genes backwards in time with real and virtual lineages. As an application, the transition functions of the non-neutral Moran and Λ-coalescent models are expressed as mixtures of the transition functions of the dual process.