Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production.

Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic.

Metabolic enzyme clustering by coiled coils improves the biosynthesis of resveratrol and mevalonate.

The clustering of biosynthetic enzymes is used in nature to channel reaction products and increase the yield of compounds produced by multiple reaction steps. The coupling of multiple enzymes has been shown to increase the biosynthetic product yield. Different clustering strategies have particular advantages as the spatial organization of multiple enzymes creates biocatalytic cascades with a higher efficiency of biochemical reaction.

Function-based mutation-resistant synthetic signaling device activated by HIV-1 proteolysis.

The high mutation rate of the human immunodeficiency virus type 1 (HIV-1) virus is a major problem since it evades the function of antibodies and chemical inhibitors. Here, we demonstrate a viral detection strategy based on synthetic biology principles to detect a specific viral function rather than a particular viral protein. The resistance caused by mutations can be circumvented since the mutations that cause the loss of function also incapacitate the virus.

A bistable genetic switch based on designable DNA-binding domains.

Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains.

Designable DNA-binding domains enable construction of logic circuits in mammalian cells.

Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA- binding domains.

Noninvasive high-throughput single-cell analysis of HIV protease activity using ratiometric flow cytometry.

To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine.

DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.

Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences.