PURE mRNA display and cDNA display provide rapid detection of core epitope motif via high-throughput sequencing.

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

Identification of conditions for efficient cell-sized liposome preparation using commercially available reconstituted in vitro transcription-translation system.

Attempts to create complex molecular systems that mimic parts of cellular systems using a bottom-up approach have become important in the field of biology. Among various molecular systems, in vitro protein synthesis inside lipid vesicles (liposomes), which we refer to as the artificial cell, has become an attractive system because it possesses two fundamental features of living cells: central dogma, and compartmentalization. Here, we investigated the effect of altering the amount or concentration of four constituents of the artificial cell consisting of a commercially available reconstituted in vitro transcription-translation (IVTT) system. As this IVTT system is available worldwide, the results will be useful to the scientific community when shared, unlike those from a lab-made IVTT system. We succeeded in revealing the effect and trend of altering each parameter and identified a suitable condition for preparing liposomes that are unilamellar and can synthesize proteins equally as well as the original IVTT system. Because the commercially available reconstituted IVTT system is an important standardization tool and the constituents can be adjusted as desired, our results will be useful for the bottom-up creation of more complex molecular systems.

Time-programmable drug dosing allows the manipulation, suppression and reversal of antibiotic drug resistance in vitro.

Multi-drug strategies have been attempted to prolong the efficacy of existing antibiotics, but with limited success. Here we show that the evolution of multi-drug-resistant Escherichia coli can be manipulated in vitro by administering pairs of antibiotics and switching between them in ON/OFF manner. Using a multiplexed cell culture system, we find that switching between certain combinations of antibiotics completely suppresses the development of resistance to one of the antibiotics. Using this data, we develop a simple deterministic model, which allows us to predict the fate of multi-drug evolution in this system. Furthermore, we are able to reverse established drug resistance based on the model prediction by modulating antibiotic selection stresses. Our results support the idea that the development of antibiotic resistance may be potentially controlled via continuous switching of drugs.