(164ad) Characterization of Mneongreen in the Cell-Free Protein Synthesis System and Optimizations of Signal Output for Biosensor Application

The cell-free system (CFS) has allowed for a new wave of innovative biosensors to emerge. Many traditional biosensors currently used suffer from inaccuracies largely due to the analyte in the sample being removed and placed in a foreign environment. The CFS allows the user to harness the checks and balances that come with the cellular environment these analytes are used to while being able to program complicated circuits to detect it. This allows the user to move away from faulty immediate signal amplification steps that result in high background noise and focus more on accurately detecting the biomarker of interest.

In this study, we increase the performance of the E. coli-based CFS to maximize the output protein signal to achieve a lower limit of detection. We first debut a new fluorescent protein, mNeonGreen (mNG), to the CFS and present characterization of its kinetics. With systemic optimization of the DNA sequence, cell-free reaction conditions, environmental contaminants, and storage additives, we could achieve a significantly brighter signal from mNG than commonly used fluorescent proteins (deGFP, x7.75; eGFP, x26.81; sfGFP, x2.64). We anticipate that our work will promote more sensitive cell-free sensor development and applied research for synthetic biology and biotechnology.