Kyriakakis, P. | Search bioRxiv

bioRxiv2025

Homology-Based Enzymatic Assembly of Modular T7 Phage Genome

Bacteriophages, viruses that infect bacteria, are pivotal in therapeutic, industrial, and bio-detection applications due to their unique ability to inject DNA into bacterial hosts and change the genetics and behavior of whole bacterial populations. Genetic engineering of phage genomes has expanded their potential applications with several established methods such as Golden Gate assembly, yeast cloning, {lambda} Red recombineering, and CRISPR-Cas systems. Here, we present a novel, efficient method to design and make synthetic bacteriophages in vitro without using restriction enzymes to allow for modular insertion of DNA fragments into the phage genome. The ability to create synthetic bacteriophages in vitro without the use of restriction enzymes allows for simpler engineering without the hassle or cloning limitations encountered when building domesticated bacteriophage genomes.

bioengineering↗

bioRxiv2023

Light-Guided Rabies Virus Tracing for Neural Circuit Analysis

Neuronal tracing methods are essential tools to understand the fundamental architecture of neural circuits and their connection to the overall functional behavior of the brain. Viral vectors used to map these transsynaptic connections are capable of cell-type-specific and directional-specific labeling of the neuronal connections. Herein, we describe a novel approach to guide the transsynaptic spreading of the Rabies Virus (RV) retrograde tracer using light. We built a Baculovirus (BV) as a helper virus to deliver all the functional components necessary and sufficient for a nontoxic RV to spread from neuron to neuron, with a light-actuated gene switch to control the RV polymerase, the L gene. This design should allow for precisely controlled polysynaptic viral tracing with minimal viral toxicity. To use this system in a highly scalable and automated manner, we built optoelectronics for controlling this system in vitro with a large field of view using an off-the-shelf CMOS sensor, OLED display panel, and microcontrollers. We describe the assembly of these genetic circuits using the uLoop DNA assembly method and a library of genetic parts designed for the uLoop system. Combining these tools provides a framework for increasing the capabilities of nontoxic tracing through multiple synapses and increasing the throughput of neural tracing using viruses.

neuroscience↗