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. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA− endA−), we synthesized 550±40 μg mL−1 of modified superfolder green fluorescent protein containing p-acetyl-l-phenylalanine. This yield was increased to ∼1300 μg mL−1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.

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. In endogenous and synthetic 5′-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA–TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

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. This assay is compatible with a wide range of fidelity-modulating conditions, and enables high-throughput analysis of sequence context effects on base substitution and single nucleotide deletion fidelity using a built-in template library. We validate this assay by comparing to previously established fidelity metrics, and use it to investigate neighboring sequence-mediated effects on fidelity for several DNA polymerases. Through these demonstrations, we establish the MagNIFi assay for robust, high-throughput analysis of DNA polymerase fidelity.

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. Y-family pols would be appealing for copying difficult DNA and incorporating non-natural dNTPs, due to their low fidelity and loose active site, but are limited by poor processivity and activity. In this study, the binding between Dbh and DNA was investigated to better understand how to rationally design enhanced processivity in a Y-family pol. Guided by structural simulation, a fused pol Sdbh with non-specific dsDNA binding protein Sso7d in the N-terminus was designed. This modification increased in vitro processivity 4-fold as compared to the wild-type Dbh. Additionally, bioinformatics was used to identify amino acid mutations that would increase stabilization of Dbh bound to DNA. The variant SdbhM76I further improved the processivity of Dbh by 10 fold. The variant SdbhKSKIP241–245RVRKS showed higher activity than Dbh on the incorporation of dCTP (correct) and dATP (incorrect) opposite the G (normal) or 8-oxoG(damaged) template base. These results demonstrate the capability to rationally design increases in pol processivity and catalytic efficiency through computational DNA binding predictions and the addition of non-specific DNA binding domains.

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 measurement, 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 biosensor state over time. However, slow signal transduction steps, due to the timescales of transcription and translation, bottleneck many sensing-DNA recording approaches. DNA polymerases (DNAPs) have been proposed as a solution to the signal transduction problem by operating as both the sensor and responder, but there is presently a lack of DNAPs with functional sensitivity to many desirable target ligands. Here, we engineer components of the Pol δ replicative polymerase complex of Saccharomyces cerevisiae to sense and respond to Ca2+, a metal cofactor relevant to numerous biological phenomena. Through domain insertion and binding site grafting to Pol δ subunits, we demonstrate functional allosteric sensitivity to Ca2+. Together, this work provides an important foundation for future efforts in developing DNAP-based biosensors.