Mark W. J. van Passel

Background: The use of in silico simulations as a basis for designing artificial biological systems (and experiments to characterize them) is one of the tangible differences between Synthetic Biology and “classical” Genetic Engineering. To this end, synthetic biologists have adopted approaches originating from the traditionally non-biological fields of Nonlinear Dynamics and Systems & Control Theory. However, due to the complex molecular interactions affecting the emergent properties of biological systems, mechanistic descriptions of even the simplest genetic circuits (transcriptional feedback oscillators, bi-stable switches) produced by these methods tend to be either oversimplified, or numerically intractable. More comprehensive and realistic models can be approximated by constructing “toy” genetic circuits that provide the experimenter with some degree of control over the transcriptional dynamics, and allow for experimental set-ups that generate reliable data reflecting the intracellular biochemical state in real time. To this end, we designed two genetic circuits (basic and tunable) capable of exhibiting synchronized oscillatory green fluorescent protein (GFP) expression in small populations of Escherichia coli cells. The functionality of the basic circuit was verified microscopically. High-level visualizations of computational simulations were analyzed to determine whether the reliability and utility of a synchronized transcriptional oscillator could be enhanced by the introduction of chemically inducible repressors. Results: Synchronized oscillations in GFP expression were repeatedly observed in chemically linked sub-populations of cells. Computational simulations predicted that the introduction of independently inducible repressors substantially broaden the range of conditions under which oscillations could occur, in addition to allowing the frequency of the oscillation to be tuned. Conclusions: The genetic circuits described here may prove to be valuable research tools for the study of synchronized transcriptional feedback loops under a variety of conditions and experimental set-ups. We further demonstrate the benefit of using abstract visualizations to discover subtle non-linear trends in complex dynamic models with large parameter spaces.

Quantifying patterns of adaptive divergence between taxa is a major goal in the comparative and evolutionary study of prokaryote genomes. When applied appropriately, the McDonald-Kreitman (MK) test is a powerful test of selection based on the relative frequency of non-synonymous and synonymous substitutions between species compared to non-synonymous and synonymous polymorphisms within species. The webserver ODoSE (Ortholog Direction of Selection Engine) allows the calculation of a novel extension of the MK test, the Direction of Selection (DoS) statistic, as well as the calculation of a weighted-average Neutrality Index (NI) statistic for the entire core genome, allowing for systematic analysis of the evolutionary forces shaping core genome divergence in prokaryotes. ODoSE is hosted in a Galaxy environment, which makes it easy to use and amenable to customization and is freely available at www.odose.nl.

Synthetic biology is an emerging field that combines molecular biology with engineering principles, which requires abstraction levels applied to a modular biological componentry. The Registry of Standard Biological Parts harbours such a repository of standardized parts, and thereby facilitates the combination of complex molecular modules to novel genetic circuits and devices. However, since finding the best parts for a pre-determined genetic design can be time consuming, we devised the Constructor, a web tool that recommends the smallest number of cloning steps for pre-designed circuits, and implements user-defined quality checks. We present the Constructor (http://www.systemsbiology.nl/the_constructor) as a constructive web tool that simplifies the in silico assembly of pre-designed gene circuitries from standard parts, reducing both planning and subsequent cloning time.

Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

We sought to assess whether the concept of relative entropy (information capacity), could aid our understanding of the process of horizontal gene transfer in microbes. We analyzed the differences in information capacity between prokaryotic chromosomes, genomic islands (GI), phages, and plasmids. Relative entropy was estimated using the Kullback-Leibler measure.

Horizontal gene transfer constitutes a powerful and innovative force in evolution, but often little is known about the actual origins of transferred genes. Sequence alignments are generally of limited use in tracking the original donor, since still only a small fraction of the total genetic diversity is thought to be uncovered. Alternatively, approaches based on similarities in the genome specific relative oligonucleotide frequencies do not require alignments. Even though the exact origins of horizontally transferred genes may still not be established using these compositional analyses, it does suggest that compositionally very similar regions are likely to have had a common origin. These analyses have shown that up to a third of large acquired gene clusters that reside in the same genome are compositionally very similar, indicative of a shared origin. This brings us closer to uncovering the original donors of horizontally transferred genes, and could help in elucidating possible regulatory interactions between previously unlinked sequences.

Microbial genomes do not merely evolve through the slow accumulation of mutations, but also, and often more dramatically, by taking up new DNA in a process called horizontal gene transfer. These innovation leaps in the acquisition of new traits can take place via the introgression of single genes, but also through the acquisition of large gene clusters, which are termed Genomic Islands. Since only a small proportion of all the DNA diversity has been sequenced, it can be hard to find the appropriate donors for acquired genes via sequence alignments from databases. In contrast, relative oligonucleotide frequencies represent a remarkably stable genomic signature in prokaryotes, which facilitates compositional comparisons as an alignment-free alternative for phylogenetic relatedness.