Recovery of Information Stored in Modified DNA with an Evolved Polymerase.

DNA is increasingly being explored as an alternative medium for digital information storage, but the potential information loss from degradation and associated issues with error during reading challenge its wide-scale implementation. To address this, we propose an atomic-scale encoding standard for DNA, where information is encoded in degradation-resistant analogues of natural nucleic acids (xNAs). To better enable this approach, we used directed evolution to create a polymerase capable of transforming 2'--methyl templates into double-stranded DNA.

A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires.

Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets.

Predicting Evolution of the Transcription Regulatory Network in a Bacteriophage.

Prediction of evolutionary trajectories has been an elusive goal, requiring a deep knowledge of underlying mechanisms that relate genotype to phenotype plus understanding how phenotype impacts organismal fitness. We tested our ability to predict molecular regulatory evolution in a bacteriophage (T7) whose RNA polymerase (RNAP) was altered to recognize a heterologous promoter differing by three nucleotides from the wild-type promoter. A mutant of wild-type T7 lacking its RNAP gene was passaged on a bacterial strain providing the novel RNAP in trans.

Cellular reagents for diagnostics and synthetic biology.

We have found that the overproduction of enzymes in bacteria followed by their lyophilization leads to 'cellular reagents' that can be directly used to carry out numerous molecular biology reactions. We demonstrate the use of cellular reagents in a variety of molecular diagnostics, such as TaqMan qPCR with no diminution in sensitivity, and in synthetic biology cornerstones such as the Gibson assembly of DNA fragments, where new plasmids can be constructed solely based on adding cellular reagents. Cellular reagents have significantly reduced complexity and cost of production, storage and implementation, features that should facilitate accessibility and use in resource-poor conditions.

Directed evolution of a synthetic phylogeny of programmable Trp repressors.

As synthetic regulatory programs expand in sophistication, an ever increasing number of biological components with predictable phenotypes is required. Regulators are often 'part mined' from a diverse, but uncharacterized, array of genomic sequences, often leading to idiosyncratic behavior. Here, we generate an entire synthetic phylogeny from the canonical allosteric transcription factor TrpR.

Identification of tumor-reactive B cells and systemic IgG in breast cancer based on clonal frequency in the sentinel lymph node.

A better understanding of antitumor immune responses is the key to advancing the field of cancer immunotherapy. Endogenous immunity in cancer patients, such as circulating anticancer antibodies or tumor-reactive B cells, has been historically yet incompletely described. Here, we demonstrate that tumor-draining (sentinel) lymph node (SN) is a rich source for tumor-reactive B cells that give rise to systemic IgG anticancer antibodies circulating in the bloodstream of breast cancer patients.

Compartmentalized partnered replication for the directed evolution of genetic parts and circuits.

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase.

Synthetic evolutionary origin of a proofreading reverse transcriptase.

Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3'- 5' exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently.

Addicting diverse bacteria to a noncanonical amino acid.

Engineered orthogonal translation systems have greatly enabled the expansion of the genetic code using noncanonical amino acids (NCAAs). However, the impact of NCAAs on organismal evolution remains unclear, in part because it is difficult to force the adoption of new genetic codes in organisms. By reengineering TEM-1 β-lactamase to be dependent on a NCAA, we maintained bacterial NCAA dependence for hundreds of generations without escape.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched.

Directed Evolution of a Panel of Orthogonal T7 RNA Polymerase Variants for in Vivo or in Vitro Synthetic Circuitry.

T7 RNA polymerase is the foundation of synthetic biological circuitry both in vivo and in vitro due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel.

Library generation by gene shuffling.

This unit describes the process of gene shuffling, also known as sexual PCR. Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primer-less PCR.

Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness.

Bioengineering advances have made it possible to fundamentally alter the genetic codes of organisms. However, the evolutionary consequences of expanding an organism's genetic code with a noncanonical amino acid are poorly understood. Here we show that bacteriophages evolved on a host that incorporates 3-iodotyrosine at the amber stop codon acquire neutral and beneficial mutations to this new amino acid in their proteins, demonstrating that an expanded genetic code increases evolvability.

Directed evolution of genetic parts and circuits by compartmentalized partnered replication.

Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function.

An in vitro autogene.

Recent technological advances have allowed development of increasingly complex systems for in vitro evolution. Here, we describe an in vitro autogene composed of a self-amplifying T7 RNA polymerase system. Functional autogene templates in cell-free lysate produce T7 RNA polymerase, which amplifies the autogene genetic information through a positive feedback architecture.

Abiotic self-replication.

The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled.