DNA nanotechnology: new adventures for an old warhorse

As the blueprint of life, the natural exploits of DNA are admirable. However, DNA should not only be viewed within a biological context. It is an elegantly simple yet functionally complex chemical polymer with properties that make it an ideal platform for engineering new nanotechnologies. Rapidly advancing synthesis and sequencing technologies are enabling novel unnatural applications for DNA beyond the realm of genetics. Here we explore the chemical biology of DNA nanotechnology for emerging applications in communication and digital data storage. Early studies of DNA as an alternative to magnetic and optical storage mediums have not only been promising, but have demonstrated the potential of DNA to revolutionize the way we interact with digital data in the future.United States. Defense Advanced Research Projects Agency (Contract FA8721-05-C-0002)National Institutes of Health (U.S.) (Grant 1R01EB017755)National Institutes of Health (U.S.) (Grant 1DP2OD008435)National Institutes of Health (U.S.) (Grant 1P50GM098792

Basic studies of baroclinic flows

A fully nonlinear 3-dimensional numerical model (GEOSIM), previously developed and validated for several cases of geophysical fluid flow, has been used to investigate the dynamical behavior of laboratory experiments of fluid flows similar to those of the Earth's atmosphere. The phenomena investigated are amplitude vacillation, and the response of the fluid system to uneven heating and cooling. The previous year's work included hysteresis in the transition between axisymmetric and wave flow. Investigation is also continuing of the flows in the Geophysical Fluid Flow Cell (GFFC), a low-gravity Spacelab experiment. Much of the effort in the past year has been spent in validation of the model under a wide range of external parameters including nonlinear flow regimes. With the implementation of a 3-dimensional upwind differencing scheme, higher spectral resolution, and a shorter time step, the model has been found capable of predicting the majority of flow regimes observed in one complete series of baroclinic annulus experiments of Pfeffer and co-workers. Detailed analysis of amplitude vacillation has revealed that the phase splitting described in the laboratory experiments occurs in some but not all cases. Through the use of animation of the models output, a vivid 3-dimensional view of the phase splitting was shown to the audience of the Southeastern Geophysical Fluid Dynamics Conference in March of this year. A study on interannual variability was made using GEOSIM with periodic variations in the thermal forcing. Thus far, the model has not predicted a chaotic behavior as observed in the experiments, although there is a sensitivity in the wavenumber selection to the initial conditions. Work on this subject, and on annulus experiments with non-axisymmetric thermal heating, will continue. The comparison of GEOSIM's predictions will result from the Spacelab 3 GFFC experiments continued over the past year, on a 'back-burner' basis. At this point, the study (in the form of a draft of a journal article) is nearly completed. The results from GEOSIM compared very well with the experiments, and the use of the model allows the demonstration of flow mechanics that were not possible with the experimental data. For example, animation of the model output shows that the forking of the spiral bands is a transient phenomenon, due to the differential east-west propagation of convection bands from different latitudes

Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas

Transcriptional regulation is central to the complex behavior of natural biological systems and synthetic gene circuits. Platforms for the scalable, tunable, and simple modulation of transcription would enable new abilities to study natural systems and implement artificial capabilities in living cells. Previous approaches to synthetic transcriptional regulation have relied on engineering DNA-binding proteins, which necessitate multistep processes for construction and optimization of function. Here, we show that the CRISPR/Cas system of Streptococcus pyogenes can be programmed to direct both activation and repression to natural and artificial eukaryotic promoters through the simple engineering of guide RNAs with base-pairing complementarity to target DNA sites. We demonstrate that the activity of CRISPR-based transcription factors (crisprTFs) can be tuned by directing multiple crisprTFs to different positions in natural promoters and by arraying multiple crisprTF-binding sites in the context of synthetic promoters in yeast and human cells. Furthermore, externally controllable regulatory modules can be engineered by layering gRNAs with small molecule-responsive proteins. Additionally, single nucleotide substitutions within promoters are sufficient to render them orthogonal with respect to the same gRNA-guided crisprTF. We envision that CRISPR-based eukaryotic gene regulation will enable the facile construction of scalable synthetic gene circuits and open up new approaches for mapping natural gene networks and their effects on complex cellular phenotypes

Synthetic mixed-signal computation in living cells

Living cells implement complex computations on the continuous environmental signals that they encounter. These computations involve both analogue- and digital-like processing of signals to give rise to complex developmental programs, context-dependent behaviours and homeostatic activities. In contrast to natural biological systems, synthetic biological systems have largely focused on either digital or analogue computation separately. Here we integrate analogue and digital computation to implement complex hybrid synthetic genetic programs in living cells. We present a framework for building comparator gene circuits to digitize analogue inputs based on different thresholds. We then demonstrate that comparators can be predictably composed together to build band-pass filters, ternary logic systems and multi-level analogue-to-digital converters. In addition, we interface these analogue-to-digital circuits with other digital gene circuits to enable concentration-dependent logic. We expect that this hybrid computational paradigm will enable new industrial, diagnostic and therapeutic applications with engineered cells.Fundacao para a Ciencia e a Tecnologia (Fellowship SFRH/BD/51576/2011)National Science Foundation (U.S.) (1350625)National Science Foundation (U.S.) (1124247)United States. Office of Naval Research (N000141310424)National Institutes of Health (U.S.) (New Innovator Award 1DP2OD008435)National Centers for Systems Biology (U.S.) (1P50GM098792

Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics

New therapeutic strategies are needed to treat infections caused by drug-resistant bacteria, which constitute a major growing threat to human health. Here, we use a high-throughput technology to identify combinatorial genetic perturbations that can enhance the killing of drug-resistant bacteria with antibiotic treatment. This strategy, Combinatorial Genetics En Masse (CombiGEM), enables the rapid generation of high-order barcoded combinations of genetic elements for high-throughput multiplexed characterization based on next-generation sequencing. We created ~34,000 pairwise combinations of Escherichia coli transcription factor (TF) overexpression constructs. Using Illumina sequencing, we identified diverse perturbations in antibiotic-resistance phenotypes against carbapenem-resistant Enterobacteriaceae. Specifically, we found multiple TF combinations that potentiated antibiotic killing by up to 10[superscript 6]-fold and delivered these combinations via phagemids to increase the killing of highly drug-resistant E. coli harboring New Delhi metallo-beta-lactamase-1. Moreover, we constructed libraries of three-wise combinations of transcription factors with >4 million unique members and demonstrated that these could be tracked via next-generation sequencing. We envision that CombiGEM could be extended to other model organisms, disease models, and phenotypes, where it could accelerate massively parallel combinatorial genetics studies for a broad range of biomedical and biotechnology applications, including the treatment of antibiotic-resistant infections.National Institutes of Health (U.S.) (New Innovator Award DP2 OD008435)United States. Office of Naval ResearchEllison Medical Foundation (New Scholar in Aging Award)Henry L. and Grace Doherty Charitable Foundatio

Programming a Human Commensal Bacterium, Bacteroides thetaiotaomicron, to Sense and Respond to Stimuli in the Murine Gut Microbiota

Engineering commensal organisms for challenging applications, such as modulating the gut ecosystem, is hampered by the lack of genetic parts. Here, we describe promoters, ribosome-binding sites, and inducible systems for use in the commensal bacterium Bacteroides thetaiotaomicron, a prevalent and stable resident of the human gut. We achieve up to 10,000-fold range in constitutive gene expression and 100-fold regulation of gene expression with inducible promoters and use these parts to record DNA-encoded memory in the genome. We use CRISPR interference (CRISPRi) for regulated knockdown of recombinant and endogenous gene expression to alter the metabolic capacity of B. thetaiotaomicron and its resistance to antimicrobial peptides. Finally, we show that inducible CRISPRi and recombinase systems can function in B. thetaiotaomicron colonizing the mouse gut. These results provide a blueprint for engineering new chassis and a resource to engineer Bacteroides for surveillance of or therapeutic delivery to the gut microbiome.National Science Foundation (U.S.) (Grant EEC-0540879)National Institutes of Health (U.S.) (Grants P50GM098792, 1DP2OD008435, 1R01EB017755, and GM095765)United States. Defense Advanced Research Projects Agency (Grant CLIO N66001-12-C-4016)United States. Defense Threat Reduction Agency (Grant HDTRA1-14-1-0007)United States. Office of Naval Research (Grant N00014-13-1-0424)Massachusetts Institute of Technology. Center for Microbiome Informatics and TherapeuticsQUALCOMM Inc. (Innovation Fellowship

Combating biofilms and antibiotic resistance using synthetic biology

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references (leaves 81-86).Bacterial infections represent a significant source of morbidity and mortality. Biofilms and antibiotic resistance pose challenges to our future ability to treat bacterial diseases with antibiotics (1). Bacteria frequently live in biofilms, which are surface-associated communities encased in a hydrated extracellular polymeric substances (EPS) matrix (2, 3). Biofilms are crucial in the pathogenesis of many clinically-important infections and are difficult to eradicate because they exhibit resistance to antimicrobial agents and removal by host immune systems (4). Antibiotics can even induce biofilm formation (5, 6). The development of antibiotic-resistant bacteria is also a growing medical problem. Antibiotic resistance genes can be acquired by horizontal gene transfer and passed vertically to later generations (7). Antibiotic resistance can also result from persistence, a phenomena in which a subpopulation of cells can withstand antibiotic treatment without containing antibiotic-resistance genes (8). These problems, coupled with decreasing output of new antibiotics, have highlighted the need for new treatments for bacterial infections (1, 9-12). I developed three novel strategies for attacking bacterial biofilms and antibiotic resistance using synthetic biology. To remove biofilms, I engineered bacteriophage to express a biofilm degrading enzyme during infection to simultaneously attack biofilm cells and the biofilm EPS matrix. These enzymatically-active bacteriophage substantially reduced biofilm cell counts by 4.5 orders of magnitude (-99.997% removal), which was about two orders of magnitude better than that of non-enzymatic phage. To address antibiotic-resistant bacteria, I targeted gene networks with synthetic bacteriophage to create antibiotic adjuvants.(cont.) Suppressing the SOS network with engineered bacteriophage enhanced killing by ofloxacin, a quinolone drug, by over 2.7 and 4.5 orders of magnitude compared with control bacteriophage plus ofloxacin and ofloxacin alone, respectively. I also built phage that targeted multiple gene networks and demonstrated their effectiveness as antibiotic adjuvants. Engineered bacteriophage reduced the number of antibiotic-resistant bacteria and performed as strong adjuvants for other bactericidal antibiotics such as aminoglycosides and P-lactams. Finally, I designed synthetic in vivo sensors for antibiotic-resistance genes that can be coupled with effector components to kill cells carrying resistance genes or to block horizontal transmission of those genes. My work demonstrates the feasibility and benefits of using engineered bacteriophage and synthetic biology constructs to address the dual threats of bacterial biofilms and antibiotic-resistant bacteria.by Timothy Kuan-Ta Lu.Ph.D

A feedback analysis of outer hair cell dynamics

Thesis (M.Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2003.Includes bibliographical references (leaves 144-146).Outer hair cells (OHCs) generate active forces in the mammalian cochlea. Acting as cochlear amplifiers, OHCs can counteract viscous drag, generating high gain at characteristic frequencies and allowing for the sharp frequency selectivity and sensitivity observed in mammals. Excitatory displacement of the basilar membrane causes depolarization of OHC membrane potentials which results in contraction. The motor protein prestin is driven by receptor potentials. However, low-pass filtering by the plasma membrane should severely attenuate the receptor potential at high frequencies (> 100 kHz) where mammalian hearing has been observed. Thus, an open question is how OHCs can respond at these high frequencies despite their low frequency cutoff. Inspired by the use of feedback in mechanical and electrical systems to accelerate slow poles, I demonstrate that negative feedback from the coupling of two mechanical modes of vibration can lead to a membrane time constant speedup and a sharpening of the mechanical response.y Timothy K. Lu.M.Eng.and S.B

Versatile and on-demand biologics co-production in yeast

Current limitations to on-demand drug manufacturing can be addressed by technologies that streamline manufacturing processes. Combining the production of two or more drugs into a single batch could not only be useful for research, clinical studies, and urgent therapies but also effective when combination therapies are needed or where resources are scarce. Here we propose strategies to concurrently produce multiple biologics from yeast in single batches by multiplexing strain development, cell culture, separation, and purification. We demonstrate proof-of-concept for three biologics co-production strategies: (i) inducible expression of multiple biologics and control over the ratio between biologic drugs produced together; (ii) consolidated bioprocessing; and (iii) co-expression and co-purification of a mixture of two monoclonal antibodies. We then use these basic strategies to produce drug mixtures as well as to separate drugs. These strategies offer a diverse array of options for on-demand, flexible, low-cost, and decentralized biomanufacturing applications without the need for specialized equipment

Complete genome sequence of pseudomonas aeruginosa Phage vB_PaeM_CEB_DP1

vB_PaeM_CEB_DP1 is a Pseudomonas aeruginosa bacteriophage (phage) belonging to the Pbunalikevirus genus of the Myoviridae family of phages. It was isolated from hospital sewage. vB_PaeM_CEB_DP1 is a double-stranded DNA (dsDNA) phage, with a genome of 66,158 bp, containing 89 predicted open reading frames.NIH -National Institutes of Health(1DP2OD008435