A general strategy for expanding polymerase function by droplet microfluidics.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics.

DNA polymerase-mediated synthesis of unbiased threose nucleic acid (TNA) polymers requires 7-deazaguanine to suppress G:G mispairing during TNA transcription.

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA.

Automated solid-phase synthesis of high capacity oligo-dT cellulose for affinity purification of poly-A tagged biomolecules.

Affinity purification of poly-adenylated biomolecules using solid supports that are derivatized with poly-thymidine oligonucleotides provides a powerful method for isolating cellular mRNA. These systems have also been used to purify mRNA-peptide fusions generated by RNA-display. However, the commercial source for high capacity oligo-dT cellulose was recently discontinued.

General approach for characterizing in vitro selected peptides with protein binding affinity.

In vitro selection technologies are important tools for identifying high affinity peptides to proteins of broad medical and biological interest. However, the technological advances that have made it possible to generate long lists of candidate peptides have far outpaced our ability to characterize the binding properties of individual peptides. Here, we describe a low cost strategy to rapidly synthesize, purify, screen, and characterize peptides for high binding affinity.

Comparative analysis of eukaryotic cell-free expression systems.

Cell-free protein synthesis (CFPS) allows researchers to rapidly generate functional proteins independent of cell culture. Although advances in eukaryotic lysates have increased the amount of protein that can be produced, the nuances of different translation systems lead to variability in protein production. To help overcome this problem, we have compared the relative yield and template requirements for three commonly used commercial cell-free translation systems: wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL).

A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells.

Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications.

Genome-wide profiling of human cap-independent translation-enhancing elements.

We report an in vitro selection strategy to identify RNA sequences that mediate cap-independent initiation of translation. This method entails mRNA display of trillions of genomic fragments, selection for initiation of translation and high-throughput deep sequencing. We identified >12,000 translation-enhancing elements (TEEs) in the human genome, generated a high-resolution map of human TEE-bearing regions (TBRs), and validated the function of a subset of sequences in vitro and in cultured cells.

Effects of sucrose and sorbitol on cement-based stabilization/solidification of toxic metal waste.

The effects of sucrose or sorbitol addition on the hydration, unconfined compressive strength and leachability of Portland cement pastes containing 1% Pb and 1% Zn were studied as a function of time. Whereas Pb and Zn were found to shorten the time to achieve maximum hydration of Portland cement, the combination of these metals with 0.15 wt% sucrose or 0.