Abstract. The genus Agalinis (commonly known as the “false foxgloves”) belonging to the family Orobanchaceae, is native to the Western Hemisphere. Approximately 40 species are distributed across temperate North America, and a large group is native to Missouri and the Midwestern United States. Twenty-one species of this genus warrant conservation measures. Our main aims were to understand the migration and diversification of temperate North American Agalinis, as well as focusing on the six species native to the state of Missouri. This study is the first to investigate the evolutionary diversification of this genus in temperate North America. Computer applications including BEAST (Bayesian evolutionary analysis sampling trees) and SDIVA (statistical dispersal vicariance analysis), and secondary calibrations from previous studies were implemented to understand the diversification timings and ancestral areas of this group. Our study points to southeastern United States as the center of diversity and place of origin of the Agalinis in temperate North America, from which they spread to the rest of the United States and even migrated to Canada, around the mid-late Miocene period. We were also able to trace the biogeography of the Missouri natives which diversified between the late Miocene and the Pleistocene period. It may be hypothesized that climatic shifts and increase in seasonality during the mid-late Miocene period was one of the primary causes leading to the migration and diversification of this genus throughout temperate North America.

Synthetic biologists construct parts, devices and systems to engineer cells for applications in medicine, biofuels, chemical commodities, and the environment. A common problem with projects that focus on the synthesis of natural and engineered proteins is unpredictable translation directed by ribosomal binding sites (RBS). Researchers have hypothesized that base pairing interactions between RBSs and downstream coding sequences form secondary structures that affect the efficiency of translation initiation. Bicistronic designs (BCDs) were developed in 2013 by Drew Endy and his colleagues to enable more predictable and reliable in vivo translation. BCDs have an RBS that leads to the production of a leader polypeptide with no functionality and a second RBS that directs the production of a protein of interest. BCDs are better at producing predictable protein levels in bacterial cells than simple RBSs, thereby improving the ability of BCDs to function reliably and predictably as classic synthetic biology parts. In a companion study, we used GFP expression to compare the translational efficiencies of 19 BCDs during cell‐free protein synthesis (CFPS) to their efficiencies in vivo and found a rank correlation of 0.88. This report describes our efforts to expand the toolkit for CFPS protein production to microfluidic droplets. We used a microfluidic flow control system to rapidly produce nanoliter‐scale droplets and characterized them with microscopy. Image analysis of the droplet fluorescence intensities allowed us to quantify and compare BCD‐directed protein synthesis within droplets. We automated this process for many images to accelerate the workflow. We focused on three BCDs for CFPS of GFP in droplets and documented significant increases in observed fluorescence compared to batch CFPS outside of droplets. We also found that the rank order function for the three BCDs was the same in droplets as it was in batch CFPS. Our results support the use of BCDs to gain predictability of protein production in CFPS. These results set the stage for partitioning large libraries of gene regulatory combinations into microfluidic droplets to find the best combinations suited for a given synthetic biology application.Support or Funding InformationNSF RUI MCB‐1613281 to Missouri Western State University, NSF RUI MCB‐1613203 to Davidson College

Synthetic biology integrates molecular biology tools and an engineering mindset to address challenges in medicine, agriculture, bioremediation, and biomanufacturing. A persistent problem in synthetic biology has been designing genetic circuits that produce predictable levels of protein. In 2013, Mutalik and colleagues developed bicistronic designs (BCDs) that make protein production more predicable in bacterial cells (in vivo). With the growing interest in producing proteins outside of cells (in vitro), we wanted to know if BCDs would work as predictably in cell-free protein synthesis (CFPS) as they do in E. coli cells. We tested 20 BCDs in CFPS and found they performed very similarly in vitro and in vivo. As a step toward developing methods for protein production in artificial cells, we also tested 3 BCDs inside nanoliter-scaled microfluidic droplets. The BCDs worked well in the microfluidic droplets, but their relative protein production levels were not as predictable as expected. These results suggest that the conditions under which gene expression happens in droplets result in a different relationship between genetic control elements such as BCDs and protein production than exists in batch CFPS or in cells. KEYWORDS: Bicistronic Design; Synthetic Biology; Cell-Free Protein Synthesis; Microfluidics

Synthetic biology takes an engineering approach to biological systems for the construction of parts, devices and systems that address challenges associated with medicine, biofuels, the environment, and more. Although the production of natural and engineered proteins is fundamental to this effort, a persistent challenge to protein production has been unreliable translation directed by a given ribosomal binding site (RBS) depending on which gene of interest is downstream of it. Based on many observations, investigators have hypothesized that the 5’ UTR containing an RBS and early codons can bind to downstream coding mRNA to form base paired secondary structures that decrease the efficiency of translation initiation. In 2013, Drew Endy and colleagues developed bicistronic designs (BCDs) for predictable and reliable in vivo translation. BCDs contain two RBSs and two protein‐encoding cistrons. The first RBS leads to the production of a 16 amino acid leader polypeptide with no functionality, whereas the second RBS directs the production of a protein encoded by a gene of interest. A key feature of BCDs is that the stop codon for the first cistron (TAA) shares its terminal adenine with the first base of the start codon (ATG) for the second cistron. In other words, TAATG is both a stop codon in one reading frame of cistron 1 and a start codon in a shifted reading frame for cistron 2. BCDs were found to be better at producing predictable protein levels in vivo than simple RBSs, thereby improving the ability of BCDs to function reliably and predictably as classic synthetic biology parts in bacterial cells. We wanted to learn if BCDs worked equally well for in vitro protein production during cell‐free protein synthesis (CFPS). CFPS employs transcriptional and translational machinery extracted from cells producing the viral T7 RNA polymerase. We built GFP expression cassettes using 19 BCDs and tested them with CFPS. We compared the rank order of the 19 BCDs in terms of in vitro CFPS GFP production with the in vivo results published by the Endy group. We calculated a rank correlation of 0.88 between the in vitro and in vivo data, showing that BCD control of protein production is comparable in both contexts. The significance of our work is that synthetic biologists using CFPS are well advised to employ BCDs so they can produce predictable amounts of protein for widespread applications in vitro.Support or Funding InformationNSF RUI MCB‐1613281 to Missouri Western State University; NSF RUI MCB‐1613203 to Davidson College