Portable, On-Demand Biomolecular Manufacturing.

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Making cellular memories.

The induction of a protracted response to a brief stimulus is a form of cellular memory. Here we describe the role of transcriptional regulation in both natural and synthetic memory networks and discuss the potential applications of engineered memory networks in medicine and industrial biotechnology.

Bioinspired Design of Artificial Signaling Systems.

Natural systems use weak interactions and avidity effects to give biological systems high specificity and signal-to-noise ratios. Here we describe design principles for engineering fusion proteins that target therapeutic fusion proteins to membrane-bound signaling receptors by first binding to designer-chosen co-receptors on the same cell surface. The key design elements are separate protein modules, one that has no signaling activity and binds to a cell surface receptor with high affinity and a second that binds to a receptor with low or moderate affinity and carries out a desired signaling or inhibitory activity. These principles are inspired by natural cytokines such as CNTF, IL-2, and IL-4 that bind strongly to nonsignaling receptors and then signal through low-affinity receptors. Such designs take advantage of the fact that when a protein is anchored to a cell membrane, its local concentration is extremely high with respect to those of other membrane proteins, so a second-step, low-affinity binding event is favored. Protein engineers have used these principles to design treatments for cancer, anemia, hypoxia, and HIV infection.

Synthetic memory circuits for tracking human cell fate.

A variety of biological phenomena, from disease progression to stem cell differentiation, are typified by a prolonged cellular response to a transient environmental cue. While biologically relevant, heterogeneity in these long-term responses is difficult to assess at the population level, necessitating the development of biological tools to track cell fate within subpopulations. Here we present a novel synthetic biology approach for identifying and tracking mammalian cell subpopulations. We constructed three genomically integrated circuits that use bistable autoregulatory transcriptional feedback to retain memory of exposure to brief stimuli. These "memory devices" are used to isolate and track the progeny of cells that responded differentially to doxycycline, hypoxia, or DNA-damaging agents. Following hypoxic or ultraviolet radiation exposure, strongly responding cells activate the memory device and exhibit changes in gene expression, growth rates, and viability for multiple generations after the initial stimulus. Taken together, these results indicate that a heritable memory of hypoxia and DNA damage exists in subpopulations that differ in long-term cell behavior.

Targeted erythropoietin selectively stimulates red blood cell expansion in vivo.

The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics.

Synthetic circuit identifies subpopulations with sustained memory of DNA damage.

Differential responses to stimuli can affect how cells succumb to disease. In yeast, DNA damage can create heterogeneous responses. To delineate how a response contributes to a cell's future behavior, we constructed a transcription-based memory circuit that detects DNA repair to isolate subpopulations with heritable damage responses. Strongly responsive cells show multigenerational effects, including growth defects and iron-associated gene expression. Less-responsive cells exhibit increased mutation frequencies but resume wild-type behavior. These two subpopulations remain distinct for multiple generations, indicating a transmissible memory of damage. Collectively, this work demonstrates the efficacy of using synthetic biology to define how environmental exposure contributes to distinct cell fates.

High prevalence of BRCA1 and BRCA2 mutations in unselected Nigerian breast cancer patients.

Inherited mutations in the BRCA1 and BRCA2 genes are the strongest genetic predictors of breast cancer and are the primary causes of familial breast/ovarian cancer syndrome. The frequency, spectrum and penetrance of mutant BRCA1/BRCA2 alleles have been determined for several populations, but little information is available for populations of African ancestry, who suffer a disproportionate burden of early onset breast cancer. We have performed complete sequence analysis of all BRCA1 and BRCA2 exons and intron-exon boundaries for 434 Nigerian breast cancer patients from the University College Hospital in Ibadan, Nigeria. In contrast to previous suggestions that BRCA1/BRCA2 mutation frequencies are low or undetectable in African American populations, we find that Nigerian breast cancer patients have an exceptionally high frequency of BRCA1 and BRCA2 mutations (7.1 and 3.9%, respectively). Sixteen different BRCA1 mutations were detected, seven of which have never been reported previously, while thirteen different BRCA2 mutations were seen, six of which were previously unreported. Thus, our data support enrichment for genetic risk factors in this relatively young cohort. To improve breast cancer outcomes, we suggest that family-based models of risk assessment and genetic counseling coupled with interventions to reduce breast cancer risk should be broadly disseminated in Nigeria and other underserved and understudied populations.

Rational engineering of an erythropoietin fusion protein to treat hypoxia.

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

A BioBrick compatible strategy for genetic modification of plants.

BACKGROUND: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. RESULTS: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. CONCLUSIONS: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.