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2014-5 Synberc Scholars

Congratulations to our graduating Synberc Scholars: Elias Valdivia, Sandy Rosales, Jacob Cota, and Ana De Oliveira! Elias will be pursuing a Ph.D. at UC Berkeley; Sandy and Jacob are both pursuing research associate positions at UC Berkeley; Ana De Oliveira will be pursuing a research associate position at the University of Washington and a possible industry internship in San Diego. Ken Groszman will be continuing on as a 2015-6 Synberc Scholar.

Elias Valdivia (Tullman-Ercek Lab, University of California, Berkeley)

Research project: We have engineered a protein pump in Salmonella to secrete proteins that can be easily recovered from the media. Heterologous proteins are made by bacteria with recombinant DNA methods for therapeutic and industrial purposes, but purification from the cell is difficult. The proteins can also aggregate to form Elias Valdiviainsoluble inclusion bodies in the cytoplasm when they are overproduced. However, many pathogenic bacteria use a type III secretion system (T3SS) to pump proteins out of the cell. In this work, we have made genomic point mutations in the prgI gene, which encodes for the T3SS needle of Salmonella enterica subsp. enterica serovar Typhimurium, that increases the secretion of heterologous proteins into the media. To determine which mutation resulted in the highest protein secretion, we compared the secreted protein titer by western blot. With genomic engineering methods, we have been able to express the T3SS mutations from the genome, enabling the protein pump to assemble properly, and 2 mutations, prgIWT::prgIP41Aand prgIWT::prgIQ48A, have increased the titer of secreted protein.

Sandy Rosales (Tullman-Ercek Lab, University of California, Berkeley)

Research project: Overproducing heterologous proteins in bacteria often results in cell toxicity and aggregation of proteins in the cytoplasm. Additionally, current procedures for purification and isolation of these cytoplasmic aggregates, or inclusion bodies, are complicated because they require refolding the protein into its native fold. Protein secretion into the extracellular space may improve the yield of native protein. We have engineered a type III secretion system (T3SS) of Salmonella by site-specific mutagenesis of the prgI gene, which encodes the needle component required for secretion, and isolated Sandy Rosalesmutations that led to higher protein titer in the culture supernatant. However, conventional western blotting techniques require a large amount of time to screen each mutant. Instead, we determined a more robust analysis of secreted enzyme by calculating Vmax, the maximum reaction rate, and kcat, which represents the turnover rate of a single enzyme. We secreted the enzyme beta-lactamase and observed that the activity of the secreted protein correlated with the amount of secreted protein titer. Through application of the rate equation, we show that a high reaction rate indicates a high concentration of protein, and a low reaction rate indicates a low concentration of protein. This enzyme assay is a more sensitive and higher-throughput method for calculating the total concentration of enzyme secreted by the prgI mutants as it requires smaller quantities and is faster than quantitative western blotting.

Jacob Cota (Tullman-Ercek Lab, University of California, Berkeley)

Research project: Bacterial microcompartments (MCPs) are bacterial organelles made up of proteins that encapsulate enzymes. We use the Pdu bacterial MCP from Salmonella enterica, which encapsulates the 1,2-propanediol utilization pathway. Our hope is to use these MCPs for green chemistry and biological synthesis. An important step towards this goal is the in vitro characterization of the interactions between lumen proteins and shell proteins of the MCPs. In order to observe these interactions we use nickel nitrilotriacetic acid column chromatography (Ni-NTA). We are using the Ni-NTA technique as a pull-down assay to determine the binding interaction between two or more proteins. The results of the Ni-NTA pull-down are then analyzed with electrophoresis, specifically SDS-PAGE, in order to determine the protein-protein interactions. The purpose of these experiments is to determine the binding of PduA, a well-characterized shell protein, and other shell proteins to lumen proteins. Our goal is to determine what enzymes interact with and bind to the compartment proteins such as PduA and how strongly they do so. We aim to tune the binding of these lumen proteins to PduA and other shell proteins. This will contribute to a tunable MCP enzyme system for heterologous proteins. 

Ken Groszman (Tabor Lab, Rice University)

Research project: The manipulation of living systems through synthetic biology will allow the scientific community to surpass many hurdles in medicine (self-regulated drug release in the gut), alternative energy (engineering metabolic pathways to produce biofuels), and manufacturing (optimized yields for chemicals and vaccines). However, before synthetic biology can tackle these larger issues, it must overcome several hurdles—namely, a lack of dynamically characterized biological parts inhibits the predictable construction of complex synthetic gene circuits.

In the past century, the electrical and control systems engineering communities leveraged a standardized and automated linear modeling technique called frequency analysis, which enables high-throughput characterization of dynamic systems. Here, we utilize frequency analysis as a modeling strategy for biological optogenetic circuits and downstream gene circuit elements. Applying sinusoidal light inputs to light-sensing systems and measuring the subsequent frequency response allows for an accurate characterization of the filtering behavior, noise, and limitations of these circuits as well as that of other genes and biological parts. This information can be compiled into a library of data sheets for biological parts which will help lift one of the great barriers between synthetic biologists and numerous novel solutions to issues ranging from medicine to alternative energy—the ability to translate complex gene circuitry into predictable expression.

Ana De Oliveira (Carothers Lab, University of Washington, Seattle)

Research project: As an experimental test-bed for investigating genetic control system design and function, the Carothers lab is engineering an E. coli-based platform to produce p-aminostyrene (p-AS), a component of advanced polymer composites. We are especially interested in using design-driven quantitative RNA device engineering to to solve control problems, such as intermediate toxicitiy, and generate high levels of production.

One of the genetic control problems is the production of a toxic intermediate, 4-amino-cinnamic acid (p-ACA), which, at certain levels, can kill the E. coli platform host. My primary research project involves the selection of RNA aptamers with high affinity and selectivitytowards the p-ACA ligand. Aptamers are usually created by selecting them from RNAs transcribed from a large random sequence DNA pool. Elsewhere aptamers have been used as platforms for therapeutics, diagnostics, drug discovery and basic research. Here, I am selecting aptamers that can be used as biosensors that recognize the presence of high p-ACA concentrations and subsequently exert control over the production pathway and expression of a transmembrane efflux pump. Therefore, these biosensors have the potential to eliminate the problem of pump over-expression and the reduction in cell-growth due to p-ACA toxicity. Professor Carothers and I hope that my aptamer selection project will provide a framework for building biosensors for more complex genetic pathways.