@article{westbrook_tang_marshall_maxwell_chappell_agrawal_dunlop_noireaux_beisel_lucks_et al._2019, title={Distinct timescales of RNA regulators enable the construction of a genetic pulse generator}, volume={116}, ISSN={["1097-0290"]}, DOI={10.1002/bit.26918}, abstractNote={AbstractTo build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher‐order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA‐based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell‐free transcription‐translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation‐based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.}, number={5}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Westbrook, Alexandra and Tang, Xun and Marshall, Ryan and Maxwell, Colin S. and Chappell, James and Agrawal, Deepak K. and Dunlop, Mary J. and Noireaux, Vincent and Beisel, Chase L. and Lucks, Julius and et al.}, year={2019}, month={May}, pages={1139–1151} }
 @article{marshall_maxwell_collins_beisel_noireaux_2017, title={Short DNA Containing chi Sites Enhances DNA Stability and Gene Expression in E-coli Cell-Free Transcription-Translation Systems}, volume={114}, ISSN={["1097-0290"]}, DOI={10.1002/bit.26333}, abstractNote={ABSTRACTEscherichia coli cell‐free transcription–translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double‐stranded DNA encoding χ sites—eight base‐pair sequences preferentially bound by the RecBCD recombination machinery—stabilizes linear DNA and greatly enhances the TXTL‐based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ‐site DNA and the DNA‐binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ‐site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137–2141. © 2017 Wiley Periodicals, Inc.}, number={9}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Marshall, Ryan and Maxwell, Colin S. and Collins, Scott P. and Beisel, Chase L. and Noireaux, Vincent}, year={2017}, month={Sep}, pages={2137–2141} }