SYNZIP Protein Interaction Toolbox: in Vitro and in Vivo Specifications of Heterospecific Coiled-Coil Interaction Domains

The synthetic biology toolkit contains a growing number of parts for regulating transcription and translation, but very few that can be used to control protein association. Here we report characterization of 22 previously published heterospecific synthetic coiled-coil peptides called SYNZIPs. We present biophysical analysis of the oligomerization states, helix orientations, and affinities of 27 SYNZIP pairs. SYNZIP pairs were also tested for interaction in two cell-based assays. In a yeast two-hybrid screen, >85% of 253 comparable interactions were consistent with prior in vitro measurements made using coiled-coil microarrays. In a yeast-signaling assay controlled by coiled-coil mediated scaffolding, 12 SYNZIP pairs were successfully used to down-regulate the expression of a reporter gene following treatment with α-factor. Characterization of these interaction modules dramatically increases the number of available protein interaction parts for synthetic biology and should facilitate a wide range of molecular engineering applications. Summary characteristics of 27 SYNZIP peptide pairs are reported in specification sheets available in the Supporting Information and at the SYNZIP Web site [http://keatingweb.mit.edu/SYNZIP/].National Science Foundation (U.S.) (NSF award MCB 0950233)National Institutes of Health (U.S.) (grant RO1 GM55040)National Institutes of Health (U.S.) (grant PN2 EY016546)National Institutes of Health (U.S.) (grant P50 GMO81879)National Science Foundation (U.S.). Synthetic Biology Engineering Research CenterHoward Hughes Medical Institut

Hyperspectral mapping of human primary and stem cells at cell–matrix interfaces

Extracellular matrices interface with cells to promote cell growth and tissue development. Given this critical role, matrix mimetics are introduced to enable biomedical materials ranging from tissue engineering scaffolds and tumor models to organoids for drug screening and implant surface coatings. Traditional microscopy methods are used to evaluate such materials in their ability to support exploitable cell responses, which are expressed in changes in cell proliferation rates and morphology. However, the physical imaging methods do not capture the chemistry of cells at cell–matrix interfaces. Herein, we report hyperspectral imaging to map the chemistry of human primary and embryonic stem cells grown on matrix materials, both native and artificial. We provide the statistical analysis of changes in lipid and protein content of the cells obtained from infrared spectral maps to conclude matrix morphologies as a major determinant of biochemical cell responses. The study demonstrates an effective methodology for evaluating bespoke matrix materials directly at cell–matrix interfaces

Differentially Instructive Extracellular Protein Micro-nets

An ability to construct biological matter from the molecule up holds promise for applications ranging from smart materials to integrated biophysical models for synthetic biology. Biomolecular self-assembly is an efficient strategy for biomaterial construction which can be programmed to support desired function. A challenge remains in replicating the strategy synthetically, that is at will, and differentially, that is for a specific function at a given length scale. Here we introduce a self-assembly topology enabling a net-like architectural mimetic of native extracellular matrices capable of differential responses to cell adhesion-enhanced mammalian cell attachment and proliferation, and enhanced resistance to bacterial colonization-at the native sub-millimeter length scales. The biological performance of such protein micro-nets directly correlates with their morphological and chemical properties, offering thus an application model for differential extracellular matrices.</p