William E. Bentley

We create molecular tools to understand the regulation of genetic curcuits during applied stresses. We also use transcriptional promoter probes, quantitative RT-PCR, Western analyses to gain near real time information on the dynamics of metabolites, genes, proteins, and protein assemblies in targeted circuits.

One objective is to alter the intracellular environment to improve cellular processes, including the production of recombinant proteins. In order to make use of the vast quantities of data, we need to organize them in reduced dimensional space, develop appropriate mathematical models, and then ultimately control phenotypic behavior. This is a component of Systems Biology. We are also actively pursuing transient metabolic controllers to minimize pleiotropy. This is a component of Synthetic Biology. We incorporate signal transduction modalities and RNA controllers to modulate, in vivo, the level of specific regulatory proteins, and downstream proteins in cascaded control loops. We are also the first group to develop a means to control bacterial subpopulations and sort quantized quorums based on QS signaling (see Servinsky et al., ISME Journal, 2015). With this technology we can answer questions like 'How many bacteria of our group should seek out new and better territories?' or 'If our entire population is challenged with an antibiotic, should we all make resistance proteins or just a few, so our colony survives in the long run?'

One exciting target is a newly characterized signal transduction pathway that communicates cell population, enabling individual bacteria to act with multicellularity. This phenomenon, also known as 'quorum sensing, (QS)', results in cell-to-cell communication and plays a significant role in regulating cell behavior. In the LuxS-mediated signaling system of E. coli, we are the first group to explore the impact of AI-2 on the transcriptome (Delisa et al., J. Bact, 2001; Wang et al., J. Bact. 2005) and we are the first group to elucidate the impact of quorum regulator, LsrR and kinase, LsrK (Li, et. al., J. Bact. 2007). We are the first group to mathematically model quorum circuitry (Li, et. al., Nature Mol. Sys. Biol., 2006). Our efforts to develop innovative controllers of signal transduction have yielded biological nanofactories that bring biochemical enzyme activities to the outer surfaces of cells so that molecules can be synthesized directly where they can be used…in our case to interrupt bacterial communication (Fernandes et al., Nature Nanotech., 2010). Current efforts include deciphering LuxS regulated genes and the impact of QS on the intracellular biomolecular landscape that influences protein synthesis; we have created a completely new system for expressing proteins that requires no operator input or sampling (Tsao et al., Met. Eng., 2010).

Finally, because we have exploited signaling pathways to control individual and groups of cells, we engineered bacteria to seek out and kill pathogens, namely, Pseudomonas aeruginosa, in the GI tract of mice. (Hwang et al., Nature Comm., 2017). This notion of synthetic biology is different than small molecule generation – the more common objective of synthetic biology. In our view, the cells themselves can be the functioning products of synthetic biology in that they can recognize, compute, actuate, and deliver, all in a programmed manner.

We are engaged in a multidisciplinary effort to create systems that serve to bridge the communication gap between biology and electronic microfabricated devices. Since biology "communicates" via small molecule signaling (like in quorum sensing above) and ion flow and because we can program devices with electrons, we have a problem in translation. We employ Nature's second most abundant biopolymer, chitosan, to serve as a "smart" stimuli-responsive interface. We are generating a biofabrication "tool box" that enables the assembly of complex biological structures onto programmable devices that allows for the accurate interrogation of the biological system. In our case, quorum sensing and bacterial communication (and even cross-Kingdom signaling) serves as a wonderful test bed for listening in on biology. We are developing all sorts of new methods for localizing DNA, proteins, cells and cell assemblies onto devices that will serve to break down complexities so that discoveries can be attributed to specific molecules, gradients, patterns, etc. With Sheref Mansy's group in Italy, we have created "artificial cells" that were able to interpret non-native chemical cues and release biological effector molecules that altered naturally growing bacterial cells. (Lentini et al., Nature Comm., 2014). We anticipate developing new tools for deciphering the presence of pathogens, for understanding and treating metabolic diseases, cancer, and hemorrhagic shock.

Finally, we have developed a means to electronically assemble and control the activity of a two-enzyme biosynthetic pathway that leads to the bacterial QS autoinducer, AI-2 (Gordonov et al., Nature Nanotech., 2014). This showed for the first time that one can electrically connect to and control biological behavior—as our signal molecule generated and controlled on chip determines behavioral phenotype at the population level. We have also for the first time, demonstrated direct electrical actuation of gene expression in bacteria. We exploited redox signaling, but actuated in a reversible manner expression of several genes in E. coli, including those that synthesize native signaling molecule, AI-1. This molecule, in turn, actuated gene expression in neighboring cells. Hence, electrically programmed cells were made to stimulate signaling via native biological means. (Tshirhart et al., Nature Comm., 2017). For a glimpse of some of these activities, please visit the Biochip Collaborative web site.