We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination. Repair of the double strand breaks and counterselection against the Landing Pad (using NiCl2 for tetA or 2-deoxy-galactose for galK) allows the isolation of modified bacteria without the use of additional antibiotic selection. We demonstrate the power of this method to make a variety of genome modifications: the exact integration, without any extraneous sequence, of the lac operon (~6.5 kbp) to any desired location in the genome and without the integration of antibiotic markers; the scarless deletion of ribosomal rrn operons (~6 kbp) through either intrachromosomal or oligonucleotide recombination; and the in situ fusion of native genes to fluorescent reporter genes without additional perturbation.

The excision and reintegration of transposable elements (TEs) restructure their host genomes, generating cellular diversity involved in evolution, development, and the etiology of human diseases. Our current knowledge of TE behavior primarily results from bulk techniques that generate time and cell ensemble averages, but cannot capture cell-to-cell variation or local environmental and temporal variability. We have developed an experimental system based on the bacterial TE IS608 that uses fluorescent reporters to directly observe single TE excision events in individual cells in real time. We find that TE activity depends upon the TE's orientation in the genome and the amount of transposase protein in the cell. We also find that TE activity is highly variable throughout the lifetime of the cell. Upon entering stationary phase, TE activity increases in cells hereditarily predisposed to TE activity. These direct observations demonstrate that real-time live-cell imaging of evolution at the molecular and individual event level is a powerful tool for the exploration of genome plasticity in stressed cells.transposable elements | evolution | quantitative biology A transposable element (TE) is a mobile genetic element that propagates within its host genome by self-catalyzed copying or excision followed by genomic reintegration (1). TEs exist in all domains of life, and the activity of TEs necessarily generates mutations in the host genome. Consequently, TEs are major contributors to disease (2-8), development (9, 10), and evolution (11, 12); they are also used as molecular tools in synthetic biology and bioengineering (13).Despite their ubiquity and importance, surprisingly little is known about the behavior and dynamics of TE activity in living cells. TE propagation rates can be inferred from comparative phylogenetic analyses of related organisms (14-20) or endpoint analyses of TE abundance within populations (11,(21)(22)(23). By making assumptions about the mechanisms of TE proliferation, models can be constructed to describe the distribution of TEs within genomes over evolutionary time scales, and sequenced genomes can be analyzed and fit to TE proliferation models to infer phylogeny of TE copies and estimate their rates of propagation (24). However, most sequencing techniques require bulk sampling of cells to provide genetic material, and sequencing is therefore generally an average over many cells. As a result, without extremely deep or single-cell sequencing techniques, most current methods are sufficient to detect only those TE events that have occurred in the germ line and therefore appear in every somatic cell in the body (25).TE rates can also be estimated by measuring relative abundances in populations that have been allowed to mutate over laboratory time scales. One of the first examples of this approach was that by Paquin and Williamson (23) to study the effects of temperature on the rate of integration of Ty retrotransposons in Saccharomyces cerevisiae after growth for 6-8 generations, resulting in yeast re...

SignificancePhylogenetic evidence suggests that a factor in the emergence of the ancestral eukaryotic cell may have been selection pressure resulting from invasion and proliferation of retroelements. Here we experimentally determine the effects of a retroelement invasion on genetically simple host organisms, and we demonstrate theoretically that the observed effects are sufficient to explain their observed rarity in bacteria. We also show that nonhomologous end-joining (NHEJ), a mechanism of DNA repair found in all extant eukaryotes, but only some bacteria, significantly enhances the efficiency of retrotransposition and the effects of retroelements on the host. We hypothesize that the interplay of NHEJ and retroelements may have played a previously unappreciated role in the evolution of advanced life.

Biological systems have long been recognized to be modular. The modular structure of the network is represented by an intrinsic parameter, modularity. Modularity is driven by the fitness of the evolving network. A number of observations show that modularity increases under increased environmental pressure. We developed a quasispecies theory for the dynamics of modularity in populations of complex networks. We show how the steady-state fitness in a randomly changing environment can be computed. We derive a relationship between rate of environmental changes and rate of growth of modularity. This relationship explains how complex networks cope with challenging environment change in order to survive and thrive.

Here, we present detailed kinetic analyses of a panel of soluble lipid kinases and phosphatases, as well as Ras activating proteins, acting on their respective membrane surface substrates. The results reveal that the mean catalytic rate of such interfacial enzymes can exhibit a strong dependence on the size of the reaction system—in this case membrane area. Experimental measurements and kinetic modeling reveal how stochastic effects stemming from low molecular copy numbers of the enzymes alter reaction kinetics based on mechanistic characteristics of the enzyme, such as positive feedback. For the competitive enzymatic cycles studied here, the final product—consisting of a specific lipid composition or Ras activity state—depends on the size of the reaction system. Furthermore, we demonstrate how these reaction size dependencies can be controlled by engineering feedback mechanisms into the enzymes.