Biology subjects

Engelhart, A. E.

Publications and source records attributed to Engelhart, A. E..

21 records · Page 2Linked to original sources

bioRxiv2020

Highly specific, multiplexed isothermal pathogen detection with fluorescent aptamer readout

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which Nucleic Acid Sequence Based Amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.

synthetic biology↗

bioRxiv2019

DNA transistors switched by the Hofmeister effect

Here we demonstrate that a DNA nanodevice can perform switchable electron transfer. The nanodevice is comprised of two strands, one of which can be selectively switched between a G-quadruplex and duplex or single-stranded conformations. In the G-quadruplex state, it binds the cofactor hemin, enabling peroxidase activity. This switching ability arises from our discovery that perchlorate, a chaotropic Hofmeister ion, selectively destabilizes duplex over G-quadruplex DNA. By varying perchlorate concentration, we show that the device can be switched between states that do and do not catalyze electron transfer catalysis. State switching can be achieved in three ways: thermally, by dilution, or by concentration. In each case, when operated in the presence of the cofactor hemin, the device catalyzes electron transfer in only the G-quadruplex state.

synthetic biology↗

bioRxiv2019

Differentiation of Pluripotent Synthetic Minimal Cells via Genetic Circuits and Programmable Mating

Synthetic minimal cells, here defined as liposomal bioreactors synthesizing protein, are a recent technology that models the intricate gene and protein networks of live cells, without the noise inherent to natural systems. Here we show a toolset for engineering combinatorial genetic circuits in synthetic cells, where distinct populations of synthetic cells control single-gene components, and those populations are assembled via programmable fusion to construct complex biological pathways. We utilize this technology to demonstrate that progenitor populations can mimic differentiation into new lineages in response to small molecule stimuli or as an effect of \"mating\" with another synthetic cell population. This provides a practical tool for metabolic engineering and natural pathway study, as well as paving the way towards the construction of live synthetic cells with complex gene pathways and the ability to form distinct lineages.

synthetic biology↗