Allen Chen

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

Engineering Living Functional Materials

Natural materials, such as bone, integrate living cells composed of organic molecules together with inorganic components. This enables combinations of functionalities, such as mechanical strength and the ability to regenerate and remodel, which are not present in existing synthetic materials. Taking a cue from nature, we propose that engineered ‘living functional materials’ and ‘living materials synthesis platforms’ that incorporate both living systems and inorganic components could transform the performance and the manufacturing of materials. As a proof-of-concept, we recently demonstrated that synthetic gene circuits in Escherichia coli enabled biofilms to be both a functional material in its own right and a materials-synthesis platform. To demonstrate the former, we engineered E. coli biofilms into a chemical-inducer-responsive electrical switch. To demonstrate the latter, we engineered E. coli biofilms to dynamically organize biotic-abiotic materials across multiple length scales, template gold nanorods, gold nanowires, and metal/semiconductor heterostructures, and synthesize semiconductor nanoparticles (Chen, A. Y. et al. (2014) Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater.13, 515–523.). Thus, tools from synthetic biology, such as those for artificial gene regulation, can be used to engineer the spatiotemporal characteristics of living systems and to interface living systems with inorganic materials. Such hybrids can possess novel properties enabled by living cells while retaining desirable functionalities of inorganic systems. These systems, as living functional materials and as living materials foundries, would provide a radically different paradigm of materials performance and synthesis–materials possessing multifunctional, self-healing, adaptable, and evolvable properties that are created and organized in a distributed, bottom-up, autonomously assembled, and environmentally sustainable manner.

SPHERES: Development of an ISS Laboratory for formation flight and docking research

The MIT Space Systems Laboratory (SSL) is developing the SPHERES (Synchronized Position Hold Engage Reorient Experimental Satellites) formation flight testbed to provide multiple investigators with a long term, replenishable, and upgradable testbed for the validation of high risk metrology, control, and autonomy technologies. These technologies are critical to the operation of distributed satellite and docking missions such as TechSat21, Starlight, Terrestrial Planet Finder, and Orbital Express. The development of SPHERES follows the guidelines set in a laboratory design philosophy created from lessons learned through the development and operation of prior microgravity testbeds by the MIT SSL. The philosophy ensures that the resulting laboratory provides a risk-tolerant and cost-effective environment that facilitates the design process and reduces the development costs of unproven technologies. The testbed consists of three free flyer units which can control their relative positions and orientations in six degrees of freedom. The testbed can operate in 2D on a laboratory platform and in 3D on NASAs KC-135 and inside the International Space Station. Flight tests aboard NASAs KC-135 and studies in the ground laboratory confirm the functionality of SPHERES.

Asymptotic parachute performance sensitivity

In 2010, the Mars Science Laboratory mission will pioneer the next generation of robotic Entry, Descent, and Landing systems by delivering the largest and most capable rover to date to the surface of Mars. In addition to landing more mass than any other mission to Mars, Mars Science Laboratory will also provide scientists with unprecedented access to regions of Mars that have been previously unreachable. By providing an Entry, Descent, and Landing system capable of landing at altitudes as high as 2 km above the reference gravitational equipotential surface, or areoid, as defined by the Mars Orbiting Laser Altimeter program, Mars Science Laboratory will demonstrate sufficient performance to land on 83% of the planets surface. By contrast, the highest altitude landing to date on Mars has been the Mars Exploration Rover at 1.3 km below the areoid. The coupling of this improved altitude performance with latitude limits as large as 60 degrees off of the equator and a precise delivery to within 10 km of a surface target, will allow the science community to select the Mars Science Laboratory landing site from thousands of scientifically interesting possibilities. In meeting these requirements, Mars Science Laboratory is extending the limits of the Entry, Descent, and Landing technologies qualified by the Mars Viking, Mars Pathfinder, and Mars Exploration Rover missions. Specifically, the drag deceleration provided by a Viking-heritage 16.15 m supersonic Disk-Gap-Band parachute in the thin atmosphere of Mars is insufficient, at the altitudes and ballistic coefficients under consideration by the Mars Science Laboratory project, to maintain necessary altitude performance and timeline margin. This paper defines and discusses the asymptotic parachute performance observed in Monte Carlo simulation and performance analysis and its effect on the Mars Science Laboratory entry, descent, and landing architecture

Synthetic Biogenesis of Bacterial Amyloid Nanomaterials with Tunable Inorganic-Organic Interfaces and Electrical Conductivity.

Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires. Specifically, we used cell-synthesized curli fibers as templates for nucleating and growing gold nanoparticles and showed that nanoparticle size could be modulated as a function of curli fiber gold-binding affinity. Furthermore, we demonstrated that gold nanoparticles can be preseeded onto curli fibers and followed by gold enhancement to form nanowires. Using these two approaches, we created artificial cellular systems that integrate inorganic-organic materials to achieve tunable electrical conductivity. We envision that cell-synthesized amyloid nanofibers will be useful for interfacing abiotic and biotic systems to create living functional materials..

Synthesizing and Patterning Tunable Multiscale Materials with Engineered Biofilms

A major challenge in materials science is to create self-assembling, functional, and environmentally responsive materials which can be patterned across multiple length scales. Natural biological systems, such as biofilms, shells, and skeletal tissues, implement dynamic regulatory programs to assemble complex multiscale materials comprised of living and non-living components1–9. Such systems can provide inspiration for the design of heterogeneous functional systems which integrate biotic and abiotic materials via hierarchical self-assembly. Here, we present a synthetic-biology platform for synthesizing and patterning self-assembled functional amyloid materials across multiple length scales with bacterial biofilms. We engineered Escherichia coli curli amyloid production under the tight control of synthetic regulatory circuits and interfaced amyloids with inorganic materials to create a biofilm-based electrical switch whose conductance can be selectively toggled by specific environmental signals. Furthermore, we externally tuned synthetic biofilms to build nanoscale amyloid biomaterials with different structure and composition through the controlled expression of their constituent subunits with artificial gene circuits. By using synthetic cell-cell communication, our engineered biofilms can also autonomously manufacture dynamic materials whose structure and composition change with time. In addition, we show that by combining subunit-level protein engineering, controlled genetic expression of self-assembling subunit proteins, and macroscale spatial gradients, synthetic biofilms can pattern protein biomaterials across multiple length scales. This work lays a foundation for synthesizing, patterning, and controlling composite materials with engineered biological systems. We envision that this approach can be expanded to other cellular and biomaterials contexts for the construction of self-organizing, environmentally responsive, and tunable multiscale composite materials with heterogeneous functionalities.