Congratulations to our new class of Synberc Scholars: Bremy Alburquerque, Marissa Dominguez, Ken Groszman (returning Scholar), Nelson Hall, Zamzam Hashi, Baylee Murphy, Michelle Reid, Katri Sofjan, and Christina Zeina. We look forward to a great year with these talented undergraduate students!
Bremy Alburquerque (Weiss Lab, MIT)
Research project: The main goal of this project is to create new tools for RNA-based therapeutic regulation. In order to improve the immunological response of current “state of the art” RNA technologies, we incorporate genetic circuit platforms from synthetic biology. RNA replicons are an alternative method to nucleic acid based vaccination commonly seen today, and possess numerous advantages over traditional DNA-based vectors or non-replicating RNA. By utilizing RNA-binding proteins, we have created novel repressors and activators capable of turning genes of interest ON and OFF. We have further developed robust methods to regulate the binding and stability of our various repressors using external control from small molecules, allowing us to create vaccines that can be switched from one state to another.
In addition, with inhibitors controlled by our small molecule regulated system, we can conditionally limit the anti-viral innate immune response involving the type 1 interferon receptor B18R which would otherwise impede the replication of our RNA replicons. With all of these implementations, the designed circuit can be used to boost antigen expression while stimulating the innate human response. These circuits would ideally help our society save time and economic resources by reducing the number of vaccines that patients around the world need to receive. Furthermore, these circuits could hopefully impact millions more as the extension of our regulatory tool box to other RNA therapeutics could expand the disease treatments available to a host of other diseases.
Marissa Dominguez (Carothers Lab, University of Washington, Seattle)
Research project: RNA biosensors implemented into existing engineered p-AS pathways would allow for gene expression modulation within E.coli bacteria. Sequences were generated and underwent folding simulations in silico, and four were tested in vitro using a fluorescence assay. Several of the devices tested showed promising results indicated by differences in fluorescence signal output that suggested dependence on the presence of pAF. We will use these results to inform new design rules for additional pAF biosensors in hopes that co-transcriptional folding trajectories can be used as a way to directly screen and engineer these devices.
Ken Groszman (Tabor Lab, Rice University) - Ken has graduated from the Synberc Scholars program - we wish him the best on his continued studies!
Research project: Expression levels in genetic circuits vary widely as a function of growth rate. This poses an important issue for synthetic biology, where variation in the extracellular context of a gene circuit may cause it to behave unpredictably or even break. Previous research has shown that constant gene expression can be achieved through negative autoregulation. The recent development of CRISPR interference (CRISPRi) as a sequence-specific repression tool enables the creation of an autoregulation module for any genetic component by co-expressing a promoter-specific single guide RNA (sgRNA) with the output gene. The efficacy of this system is confirmed through a computational model and in vivo characterization in Escherichia coli is underway. Growth-rate independent genetic circuits will enable more reliable behavior from genetic circuits in changing conditions within an experiment, from-lab-to-lab, and on the field.
Nelson Hall (Weiss Lab, MIT)
Research project: There exist a number of well-characterized platforms for engineering intercellular communication in bacteria and yeast, but engineering intercellular communication in mammalian cells remains a challenge. Luckily, almost all mammalian cell types secrete small virus-sized extracellular vesicles called exosomes, which are thought to be another method by which cells can communicate by transferring biomolecules (miRNA, proteins, mRNA, dsDNA) from cell to cell. We are currently engineering exosomes to selectively package and transmit proteins and RNAs of interest by modifying well-characterized exosomal marker proteins to either bind and carry important RNAs or to carry with it a transcription factor of interest with a cleavable peptide linker. The platform has the advantage of both specificity and targeting. By using various protease-linker pairs we ensure that only the appropriate recipient cell with the appropriate protease is able to cleave and react to a given exosomal cargo. And by modifying the exterior of the exosomes to contain various ligand-binding domains we may be able to preferentially target a specified recipient cell. Our current efforts are focused on generating an ensemble of different fusion peptides to test each of these proposed properties and characterizing the contents of exosomes generated from sender cells transfected with our cargo-carrying exosomal peptide fusions.
Baylee Murphy (Haynes Lab, Arizona State University)
Research project: It is imperative that new, high-tech advances reach all people who can benefit rather than a select few. The lab of Dr. Karmella Haynes at ASU (Tempe Campus) uses synthetic biology to discover new treatments for cancer. Certain cancers have disproportionately high mortality rates for groups who arehistorically alienated from advanced medical care and are under represented (as scientists) in cutting edge research science. Synthetic Biology aims to develop standards for biology so that biotechnology can be more accessible to creative problem-solvers from any background. In order to train young scientists to advance this goal, the project includes components in (1) classroom education, (2) wet
lab research in epigenetic engineering of cancer cells, and (3) research and activities in societal impacts. Classroom education: the trainees will structure an ASU BME100 Lab exercise around a cancer-related mutation of their choice. Wet lab: the trainee will purify plasmids that express an RFP-tagged protein that regulates epigenetics in cancer cells and contribute validation data (flow cytometry) for this protein to an open database (JBEI ICE and SB.ASU). Societal impacts: the trainees will collect facts on cancer disparities and present these plus their classroom and wet lab work with a community audience that represents an under served community.
Michelle Reid (Tullman-Ercek Lab, University of California, Berkeley)
Research project: The type III secretion system (T3SS) pathogenicity island 1 (SPI-1) present in Salmonella enterica can be used to secrete proteins into the culture media. I hypothesize that by eliminating native flagellar and T3SS virulence effector genes, we can further increase secreted protein titer of the desired protein while simultaneously minimizing secreted flagellar proteins and virulence effectors into the media, increasing purity of the protein product. We expect the results of this effort to create a strain for highly specific export of heterologous proteins into the media at levels meeting industrial demand.
Katri Sofjan (Tabor Lab, Rice University)
Research project: Two-component signal transduction systems (TCSs) allow bacteria sense and respond to environmental stimuli. Due to the diversity of detectable inputs, possible output genes, and structural families of response regulators, no streamlined and efficient method for characterizing TCSs has been developed. Characterizing these systems would build the array of biological tools available for use in research, medical, and industrial environments. TCSs are comprised of histidine kinases and their cognate response regulators. Response regulators consist of two modular domains – a receiver (REC) domain, which is phosphorylated by the kinase, and a family-specific DNA binding domain (DBD). Due to the response regulator’s modularity, TCSs can be rewired by creating chimeric response regulators which have REC and DBDs from different response regulators. This allows for characterization of unknown TCSs by connecting their input system to a known DBD and engineered promoter with high dynamic range. To accomplish this, we identify response regulators from different families, optimize their outputs through promoter engineering, and create libraries of novel chimeric proteins. This work will allow high-throughput characterization of TCSs and engineering of novel, highly optimized signaling mechanisms.
Christina Zeina (Church Lab, Harvard University)
Research project: This project utilizes phage-assisted directed evolution (PACE) for the evolution of new and novel protein functions. Developed in Professor David Liu’s lab, PACE enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in E. coli. Evolving genes are are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. As opposed to traditional directed evolution, many rounds of evolution can be carried out in a single day in a continuous manner. We have created a number of selections utilizing PACE to optimize and modify proteins of interest.