Rapid and tunable post-translational coupling of genetic circuits.

One promise of synthetic biology is the creation of genetic circuitry that enables the execution of logical programming in living cells. Such 'wet programming' is positioned to transform a wide and diverse swathe of biotechnology ranging from therapeutics and diagnostics to water treatment strategies. Although progress in the development of a library of genetic modules continues apace, a major challenge for their integration into larger circuits is the generation of sufficiently fast and precise communication between modules. An attractive approach is to integrate engineered circuits with host processes that facilitate robust cellular signalling. In this context, recent studies have demonstrated that bacterial protein degradation can trigger a precise response to stress by overloading a limited supply of intracellular proteases. Here we use protease competition to engineer rapid and tunable coupling of genetic circuits across multiple spatial and temporal scales. We characterize coupling delay times that are more than an order of magnitude faster than standard transcription-factor-based coupling methods (less than 1 min compared with ∼20-40 min) and demonstrate tunability through manipulation of the linker between the protein and its degradation tag. We use this mechanism as a platform to couple genetic clocks at the intracellular and colony level, then synchronize the multi-colony dynamics to reduce variability in both clocks. We show how the coupled clock network can be used to encode independent environmental inputs into a single time series output, thus enabling frequency multiplexing (information transmitted on a common channel by distinct frequencies) in a genetic circuit context. Our results establish a general framework for the rapid and tunable coupling of genetic circuits through the use of native 'queueing' processes such as competitive protein degradation.

A sensing array of radically coupled genetic 'biopixels'.

Although there has been considerable progress in the development of engineering principles for synthetic biology, a substantial challenge is the construction of robust circuits in a noisy cellular environment. Such an environment leads to considerable intercellular variability in circuit behaviour, which can hinder functionality at the colony level. Here we engineer the synchronization of thousands of oscillating colony 'biopixels' over centimetre-length scales through the use of synergistic intercellular coupling involving quorum sensing within a colony and gas-phase redox signalling between colonies. We use this platform to construct a liquid crystal display (LCD)-like macroscopic clock that can be used to sense arsenic via modulation of the oscillatory period. Given the repertoire of sensing capabilities of bacteria such as Escherichia coli, the ability to coordinate their behaviour over large length scales sets the stage for the construction of low cost genetic biosensors that are capable of detecting heavy metals and pathogens in the field.

A new method for vitrifying samples for cryoEM.

Almost every aspect of cryo electron microscopy (cryoEM) has been automated over the last few decades. One of the challenges that remains to be addressed is the robust and reliable preparation of vitrified specimens of suitable ice thickness. We present results from a new device for preparing vitrified samples. The successful use of the device is coupled to a new "self-blotting" grid that we have developed to provide a method for spreading a sample to a thin film without the use of externally applied filter paper. This new approach has the advantage of using small amounts of protein material, resulting in large areas of ice of a well defined thickness containing evenly distributed single particles. We believe that these methods will in the future result in a system for vitrifying grids that is completely automated.

Multiplexed TEM Specimen Preparation and Analysis of Plasmonic Nanoparticles.

We describe a system for rapidly screening hundreds of nanoparticle samples using transmission electron microscopy (TEM). The system uses a liquid handling robot to place up to 96 individual samples onto a single standard TEM grid at separate locations. The grid is then transferred into the TEM and automated software is used to acquire multiscale images of each sample. The images are then analyzed to extract metrics on the size, shape, and morphology of the nanoparticles. The system has been used to characterize plasmonically active nanomaterials.

Interfacing gene circuits with microelectronics through engineered population dynamics.

While there has been impressive progress connecting bacterial behavior with electrodes, an attractive observation to facilitate advances in synthetic biology is that the growth of a bacterial colony can be determined from impedance changes over time. Here, we interface synthetic biology with microelectronics through engineered population dynamics that regulate the accumulation of charged metabolites. We demonstrate electrical detection of the bacterial response to heavy metals via a population control circuit. We then implement this approach to a synchronized genetic oscillator where we obtain an oscillatory impedance profile from engineered bacteria. We lastly miniaturize an array of electrodes to form "bacterial integrated circuits" and demonstrate its applicability as an interface with genetic circuits. This approach paves the way for new advances in synthetic biology, analytical chemistry, and microelectronic technologies.

High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking.

We have developed a method combining microfluidics, time-lapsed single-molecule microscopy and automated image analysis allowing for the observation of an excess of 3000 complete cell cycles of exponentially growing Escherichia coli cells per experiment. The method makes it possible to analyse the rate of gene expression at the level of single proteins over the bacterial cell cycle. We also demonstrate that it is possible to count the number of non-specifically DNA binding LacI-Venus molecules using short excitation light pulses. The transcription factors are localized on the nucleoids in the cell and appear to be uniformly distributed on chromosomal DNA. An increase in the expression of LacI is observed at the beginning of the cell cycle, possibly because some gene copies are de-repressed as a result of partitioning inequalities at cell division. Finally, a size-growth rate uncertainty relation is observed where cells living in rich media vary more in the length at birth than in generation time, and the opposite is true for cells living in poorer media.

Measuring competitive fitness in dynamic environments.

Most yeast genes are dispensable for optimal growth in laboratory cultures. However, this apparent lack of fitness contribution is difficult to reconcile with the theory of natural selection. Here we use stochastic modeling to show that environmental fluctuations can select for a genetic mechanism that does not affect growth in static laboratory environments. We then present a novel experimental platform for measuring the fitness levels of specific genotypes in fluctuating environments. We test this platform by monitoring a mixed culture of two yeast strains that differ in their ability to respond to changes in carbon source yet exhibit the same fitness level in static conditions. When the sugar in the growth medium was switched between galactose and glucose, the wild-type strain gained a growth advantage over the mutant strain. Interestingly, both our computational and experimental results show that the strength of the adaptive advantage conveyed by the wild-type genotype depends on the total number of carbon source switches, not on the frequency of these fluctuations. Our results illustrate the selective power of environmental fluctuations on seemingly slight phenotypic differences in cellular response dynamics and underscore the importance of dynamic processes in the evolution of species.