DNA Assembly in 3D Printed Fluidics.

The process of connecting genetic parts-DNA assembly-is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to $5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (ßTCP) via Fusion to a High Affinity, Multimeric ßTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors.

Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival proliferation, and osteogenic differentiation of the marrow-resident stem and progenitor cells with osteogenic potential can be limited in large defects by the inflammatory microenvironment. Previous studies using EGF tethered to synthetic polymer substrates have demonstrated that surface-tethered EGF can protect human bone marrow-derived osteogenic stem and progenitor cells from pro-death inflammatory cues and enhance their proliferation without detriment to subsequent osteogenic differentiation. The objective of this study was to identify a facile means of tethering EGF to clinically-relevant ßTCP scaffolds and to demonstrate the bioactivity of EGF tethered to ßTCP using stimulation of the proliferative response of human bone-marrow derived mesenchymal stem cells (hBMSC) as a phenotypic metric. We used a phage display library and panned against ßTCP and composites of ßTCP with a degradable polyester biomaterial, together with orthogonal blocking schemes, to identify a 12-amino acid consensus binding peptide sequence, LLADTTHHRPWT, with high affinity for ßTCP. When a single copy of this ßTCP-binding peptide sequence was fused to EGF via a flexible peptide tether domain and expressed recombinantly in E. coli together with a maltose-binding domain to aid purification, the resulting fusion protein exhibited modest affinity for ßTCP. However, a fusion protein containing a linear concatamer containing 10 repeats of the binding motif the resulting fusion protein showed high affinity stable binding to ßTCP, with only 25% of the protein released after 7 days at 37oC. The fusion protein was bioactive, as assessed by its abilities to activate kinase signaling pathways downstream of the EGF receptor when presented in soluble form, and to enhance the proliferation of hBMSC when presented in tethered form on commercial ßTCP bone regeneration scaffolds.

Practical aspects of hydrophobic polycationic bactericidal "paints".

We previously discovered that coating solid surfaces with long-chained linear N-dodecyl,N-methyl-polyethylenimine makes them bactericidal and virucidal. In the present study, focusing on the use of this microbicidal paint to kill airborne Escherichia coli and Staphylococcus aureus, we have systematically investigated the dependence of this effect on the concentration and mode of application of the hydrophobic polycation, the number of coats, the nature of the solvent, and the presence of a dye in such paint. In addition, the latter's ability to be regenerated after use, stability upon repeated washings, and mammalian toxicity has been evaluated.