Engineering Ca-Dependent DNA Polymerase Activity.

Advancements in synthetic biology have provided new opportunities in biosensing, with applications ranging from genetic programming to diagnostics. Next generation biosensors aim to expand the number of accessible environments for measurements, increase the number of measurable phenomena, and improve the quality of the measurement. To this end, an emerging area in the field has been the integration of DNA as an information storage medium within biosensor outputs, leveraging nucleic acids to record the biosensor state over time.

High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing.

DNA polymerase fidelity is affected by both intrinsic properties and environmental conditions. Current strategies for measuring DNA polymerase error rate in vitro are constrained by low error subtype sensitivity, poor scalability, and lack of flexibility in types of sequence contexts that can be tested. We have developed the Magnification via Nucleotide Imbalance Fidelity (MagNIFi) assay, a scalable next-generation sequencing assay that uses a biased deoxynucleotide pool to quantitatively shift error rates into a range where errors are frequent and hence measurement is robust, while still allowing for accurate mapping to error rates under typical conditions.

DNA binding strength increases the processivity and activity of a Y-Family DNA polymerase.

DNA polymerase (pol) processivity, i.e., the bases a polymerase extends before falling off the DNA, and activity are important for copying difficult DNA sequences, including simple repeats.

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance.

Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.