A semisynthetic organism engineered for the stable expansion of the genetic alphabet.

All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP.

Selective arylthiolane deprotection by singlet oxygen: a promising tool for sensors and prodrugs.

A routine thioketal protecting group reacts rapidly and selectively with singlet oxygen to reveal ketone products in good (aryl 1,3-dithiolane) to excellent (aryl 1,3-oxathiolane) yields. Arylthiolanes are stable to biologically relevant reactive oxygen species and can be used as a light-activated gating mechanism for activating fluorescent sensors or small molecule prodrugs.

Remote control of tissue interactions via engineered photo-switchable cell surfaces.

We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand.

Cell division orientation on biospecific peptide gradients.

An assay was developed for determining cell division orientation on gradients. The methodology is based on permeating microfluidic devices with alkanethiols and subsequent printing of cell adhesive peptide gradient self-assembled monolayers (SAMs) for examining oriented cell divisions. To our knowledge, there has been no study examining the correlation between cell division orientations based on an underlying ligand gradient.

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains.

Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position.

Redox-switchable surface for controlling peptide structure.

A general surface chemistry strategy is described for the development of a new switchable material. The method modulates a surface-immobilized-molecules structure by using two orthogonal "click" reactions based on Huisgen cycloaddition and oxime chemistry, where the oxime linkage is redox active and switchable. We demonstrate this strategy by developing a noninvasive, biocompatible, in situ surface chemistry that is able to modulate the affinity of a cell-adhesive peptide to cell integrin receptors to study dynamic cell adhesion and cell migration in real time and as a new hide-and-reveal strategy for application in new types of smart biofouling biomaterials.

Microfluidic permeation printing of self-assembled monolayer gradients on surfaces for chemoselective ligand immobilization applied to cell adhesion and polarization.

To study complex cell behavior on model surfaces requires biospecific interactions between the interfacing cell and material. Developing strategies to pattern well-defined molecular gradients on surfaces is difficult but critical for studying cell adhesion, polarization, and directed cell migration. We introduce a new strategy, microfluidic SPREAD (Solute PeRmeation Enhancement And Diffusion) for inking poly(dimethylsiloxane) (PDMS) microfluidic cassettes with a gradient of alkanethiol.

Electrochemical and chemical microfluidic gold etching to generate patterned and gradient substrates for cell adhesion and cell migration.

To generate patterned substrates of self-assembled monolayers (SAMs) for cell adhesion and migration studies, a variety of gold/glass hybrid substrates were fabricated from gold evaporated on glass. A variety of surfaces were generated including gradients of gold height, completely etched gold/glass hybrids, and partially etched gold surfaces for pattern visualization. Etch rates were controlled by the alkanethiol present on the surface.

Expedient generation of patterned surface aldehydes by microfluidic oxidation for chemoselective immobilization of ligands and cells.

An expedient and inexpensive method to generate patterned aldehydes on self-assembled monolayers (SAMs) of alkanethiolates on gold with control of density for subsequent chemoselective immobilization from commercially available starting materials has been developed. Utilizing microfluidic cassettes, primary alcohol oxidation of tetra(ethylene glycol) undecane thiol and 11-mercapto-1-undecanol SAMs was performed directly on the surface generating patterned aldehyde groups with pyridinium chlorochromate. The precise density of surface aldehydes generated can be controlled and characterized by electrochemistry.

Microfluidic etching and oxime-based tailoring of biodegradable polyketoesters.

A straightforward, flexible, and inexpensive method to etch biodegradable poly(1,2,6-hexanetriol alpha-ketoglutarate) films is reported. Microfluidic delivery of the etchant, a solution of NaOH, can create micron-scale channels through local hydrolysis of the polyester film. In addition, the presence of a ketone in the repeat unit allows for prior or post chemoselective modifications, enabling the design of functionalized microchannels.

Microfluidic lithography of SAMs on gold to create dynamic surfaces for directed cell migration and contiguous cell cocultures.

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip.