(157ag) Tuning the Cell-Free Protein Synthesis System for the Development of Highly Sensitive Biosensors

The cell-free protein synthesis (CFPS) system has emerged as a powerful platform for advancing the ability to study and expand the potential of applied biotechnology. The unique open environment of the CFPS system has allowed for an unprecedented level of freedom to control and optimize biological systems and focus production on compound of interest rather than microbial growth, making this in vitro platform optimal for sensing. Many gene circuit sensors have been designed using the cell-free system platform, but most employ a highly robust bacteriophage T7 RNA polymerase (T7 RNAP). Unfortunately, using T7 RNAP for circuits puts a limit to how many ways the user can modulate the circuit because it does not require many elements to regulate its expression, which opens opportunities for circuit leak. Using the endogenous Escherichia coli RNA polymerase (Ec RNAP) can open another level of gene regulation for circuit design due to the vast number of sigma factors and cognate promoter sequences that can be chosen from, but they must be optimized to maximize the sensitivity and output signal of the sensor. Here we employ a highly sensitive and robust sigma factor to complete the endogenous RNA polymerase holoenzyme complex. To optimize further, a strong fluorescent protein is necessary to acquire data from low concentration analyte or not fully purified samples. Since the 70s, Green fluorescent protein (GFP) and its variants have been used as markers of gene and protein expression, localization, and interactions, along with sensing biomarkers and assessing the quality of protein translational systems. Recently, a new fluorescent protein was discovered and has been touted to be the brightest green/yellow fluorescent protein yet to be described. Since cell-free sensing experiments have yet to express this new fluorescent protein, in this study, we compared it to the commonly used sfGFP for the first time, to find the brightest and most robust fluorescent protein expressed in the Escherichia coli CFPS system. The fluorescent proteins were expressed and characterized by 1) pH tolerance, 2) reaction time and temperature, 3) tolerance to matrix effects from impure samples, 4) maturation time, and 5) brightness. By combining all of these advantages, the toolbox for genetic circuitry was greatly increased. This platform as a whole can be used to create sophisticated synthetic gene circuitry networks to increase the amount of data obtained from one sample source.