I was asked to write a blog post about my research that included a personal element for the BEACON Center for the Study of Evolution in Action website, then make the same post here. Hopefully other UW researchers who work on synthetic biology will post something about their research so that we know what everyone is working on.
Synthetic biology is a relatively new field that uses engineering principles to design and build novel biological functions and systems. In 2000, Michael Elowitz and colleagues constructed the first synthetic gene network called the repressilator. This genetic circuit consisted of three repressor genes connected in a feedback loop, such that each gene represses the next gene in the loop, like a genetic version of the paper-rock-scissors game. The output of the circuit was Green Fluorescent Protein (GFP) to read out the oscillatory behavior of the network using fluorescence microscopy.
Fast forward to 2011. Now we have several synthetic biology labs worldwide and hundreds of undergraduate teams that compete every year in the International Genetically Engineered Machine competition (iGEM) to engineer novel organisms. At the beginning of the summer, the students are given a kit of plasmids that encode standardized biological parts such as promoters, ribosome binding sites, coding sequences, and transcriptional terminators. These parts, calledBioBricks, have been used to engineer bacteria to function as a black and white photographic film, generate colored pigments, smell like bananas, and develop a designer vaccine for Helicobacter pylori (the bacterium that causes ulcers). Successes in synthetic biology groups include the overproduction of an anti-malaria compound, multicellular pattern formation, Craig Venter‘s synthetic cell, and the development of various biofuels.
Besides its numerous applications, synthetic biology is also a powerful system for studying evolution. After finishing my graduate work doing experimental evolution in Richard Lenski‘s lab, I was intrigued by the possibility of being able to assemble large numbers of BioBricks together on plasmids and watching how these modular DNA sequences change over time. I decided that my project should tackle one of the biggest problems in synthetic biology, evolutionary stability of genetic circuits, while at the same time involve the study of evolution. So my research deals with understanding the evolutionary stability dynamics and loss-of-function mutations in genetic circuits, then using this information to engineer mutationally robust circuits.
Genetic circuits are destined to fail unless they impart some beneficial function to the cell or there is a selective environment designed for maintaining circuit function over evolutionary time. Due to the metabolic load of having to express foreign genes, as cells divide, one with a mutant plasmid may grow slighly faster than cells having all functional plasmids. As plasmids segregate to daughter cells, a new cell may have multiple copies of the mutant plasmid and grow even faster. Eventually a cell emerges with no copies of the original plasmid and this cell can outcompete the functional cells in the population.
For my project (referenced below), I measured the stability of several BioBrick-assembled genetic circuits propagated in Escherichia coli over multiple generations and found the mutations that caused their loss-of-function. In this post, I will focus on discussing the results of one circuit called T9002. T9002 works by expressing an activator protein called LuxR. When the inducer molecule AHL is added to the media, it binds to LuxR and activates GFP expression downstream from theluxR promoter. This circuit loses function in less than 20 generations and the mutation that causes its loss-of-function is a deletion between two repeated transcriptional terminators. To measure the effect between transcriptional terminator sequence similarity and evolutionary stability, six versions of T9002 were re-engineered with a different transcriptional terminator at the end of the circuit. The figure below shows the BioBrick ID numbers/names with promoters (arrows), ribosome binding sites (ovals), coding sequences (arrows), and double transcriptional terminators (octagons) for the original circuit (top) and re-engineered circuit (bottom).
Can mutational robustness be increased by removing the sequence similarity between the first and second terminators? How predictable are mutations in genetic circuits?
The figure below shows the evolutionary stability dynamics of the original T9002 circuit vs. three of the re-engineered circuits. The circuit expression level (shown in fluorescence/OD ) is plotted vs. generations. It turns out that changing the terminator of the circuit also changes its expression level since terminator strength can change RNA degradation. The table in the corner shows the relationship of each circuit to expression level and sequence similarity between terminators. The T9002 circuit, with high sequence similarity between terminators and high expression, loses function in less than 20 generations. The T9002-E circuit has an increased evolutionary half-life of over 2-fold on average and this is likely due to having no terminator sequence similarity since its expression level is similar to T9002. The T9002-F circuit is the most robust, with a 17-fold increase in evolutionary half-life, due to having both a low expression level and low sequence similarity. The T9002-D circuit, with medium expression level and sequence similarity, has a half-life in between the T9002 and T9002-F circuits. After noticing the pattern between expression level and evolutionary half-life, I tested this relationship with several circuits and found that on average half-life decreases exponentially with expression level.
To understand the predictability of mutations in these circuits, the loss-of-function mutations in one clone from nine populations were discovered and shown in the table below. When there are repeated terminators, as in the T9002 circuit, the same exact deletion is found between terminators in all nine populations. However, when sequence similarity is decreased, as in the T9002-D circuit, there are sometimes deletions between repeated sequences, but also less predictable mutations. The T9002-E and F circuits, with no sequence similarity between terminators, have mutations of various types that involve inactivating either the luxR gene or luxRpromoter.
Overall, the T9002 circuit can be re-engineered to be more mutationally robust by decreasing its expression level and sequence similarity between terminators. Presumably deletions between repeated terminators occur at a high rate and therefore this circuit can be re-engineered to have a lower mutation rate by removing a certain class of mutations from occurring. Although decreasing mutation rate effectively increases mutational robustness (T9002 vs. T9002-E), decreasing expression level has a stronger relative effect on evolutionary half-life (T9002 and T9002-E vs. T9002-F).
My current BEACON project extends this work by understanding mutational robustness in metabolic pathways. For this project, instead of rationally re-engineering pathways, I will be using a directed evolution approach to identify designs that are the most robust.
References: Sleight S.C., B.A. Bartley, J.A. Lieviant, and H.M. Sauro. Designing and engineering evolutionary robust genetic circuits. 2010. Journal of Biological Engineering, 4:12.
iGEM 2010: Antibiotics for the 21st Century
December 29, 2010
by Michal Galdzicki
This year the UW team won the Best Health and Medicine Project of the year prize at the 2010 iGEM Jamboree. The award winning project is titled “Antibiotics for the 21st Century“. New antibiotics such as the two examples developed by this years team, will be critical in medicine for the next century. The spread of antibiotic resistance in pathogenic strains has rendered more and more long standing anti-bacterial therapeutics ineffective. The emergence of these antibiotic resistant bacteria has been largely caused by the unfettered application of broad-spectrum antibiotics in cases when such therapies were unwarranted.
At the same time, throughout the 20th century the use of these antibiotics revolutionized medicine and saved countless lives. The work that the University of Washington undergraduates performed this summer has the potential to impact the lives of millions of people in need of new antibiotics. With this motivation in mind the team from set out on their summer research project and succeeded to not only develop two new antibiotic therapy candidates, but most importantly experienced real research from start to finish.
It is the story of how these young and inexperienced, but wildly motivated students could accomplish such a feat in just a few months, which amazed me. The 2010 iGEM year at UW started with interested students coming to meetings and talks in the Winter quarter of 2010 and by the end of the summer their project evolved into a well defined biological engineering endeavor. The students this year were from several different majors, including Biology, Biochemistry, and Microbiology. Some were college freshmen, having just finished high school at the beginning of the summer, while others had worked in research laboratories before, and one student was a returning member of the gold medal decorated 2009 UW iGEM team. The interesting talks by host faculty, graduate students and invited external experts about a broad range of topics gave students background information and an overview of iGEM. For example: Dr. Rob Carlson, who is a consultant, independent researcher, entrepreneur, and iGEM judge introduced Synthetic Biology and iGEM; Dr. Marina Kalyuzhnaya, faculty from UW Microbiology, inspired the team with a explanation of metabolic engineering of methylotrpohs. Dr. Lee Pang, from the Institute for Systems Biology explained the importance of predictive modeling for bacterial re-engineering. The sponsoring faculty and their PhD students also presented about their research and then the team was ready to brainstorm ideas for this year’s iGEM project. The students were advised by PhD students, Ingrid Swanson, Justin Siegal, Matt Smith, Rob Egbert, and myself. Throughout the whole process the students worked under the guidance and oversight of professors David Baker, Herbert Sauro, Joseph Mougous, and Eric Klavins.
To explore the potential for several of the brainstormed ideas the team was organized by student interest, into sub-groups for the Spring quarter to allow students to takes their ideas and really make them into putative research plans for the summer. These students then worked within the labs of each of the faculty, relying on the expertise in those labs, to form an understanding of the motivations, the strategies, and identify pitfalls they may encounter. Every week the whole team would come together and report on the exciting plans for the summer project. Then as the Spring quarter was wrapping up, several of the intial ideas boiled to the top as exciting and feasible. Once these few best ideas rose to the top the students voted and chose to engage in the research full time for the summer academic break. The students quickly got up to speed on lab protocols, programming, and data analysis, or more emphatically, they hit the ground running. Safely, of course.
The summer brought long days and sometimes nights in the lab. Students learned to design and model proteins, clone genes, plan and assemble genetic constructs, and how to verify everything along the way. PCR reactions don’t always work and code doesn’t compile. The iGEM team learned to celebrate every little success, or sometimes even the confused frustration. But they were able to celebrate enough times to develop Antibiotics for the 21st Century. The research process is grueling no matter what the kit advertisement says and interpreting failures is an incredibly important part of the learning process. The most important goal of the iGEM experience is for the students to have fun and to learn how research is done. When they completed this project the students were proud. They succeeded and published their work on the web for everyone to see.
The project wiki, is a key component of iGEM research presentations as it details all of the scientific material and describes the work done. Putting the wiki together, from design to polished dynamic document was a feat in itself. Such polished and detailed web publication of your research work is something even most post graduate researchers don’t do. It’s extremely important for the institutional memory of the iGEM competition. Each year, new students are able to get ideas and even re-use the DNA parts created by all teams in prior years. Once complete they took their results to the iGEM Jamboree to present to their peers from around the world.
Our undergraduate students presented their project on developing two new antibiotic therapies. They were awarded a goldmedal recognizing their the high quality of their research, documentation (on the wiki), and for contributing tools that will aid future synthetic biology efforts. The 128 teams from around the world were tough competitors, including some ground breaking projects from other universities. But our students stood out, the only other US team to win their science track was MIT. We even received an iGEM trophy for the UW to show off .
Cross your fingers for the 2011 UW iGEM team!
Genome engineering workshop
December 13, 2010
The Northwest Genome Engineering Consortium workshop was held on November 16th, 2010 at the UW Medicine – South Lake Union facility in Seattle, WA. 140 attendees over the entire day enjoyed two Keynote addresses by Dr. Bruce Torbett and Dr. Toni Cathomen. In addition, we had twelve short talks and 43 posters. Eric Klavins was one of the speakers along with Farren Issacs who spoke about Genome Engineering Technologies for Rapid Programming & Evolution of Cells.
BEACON Center for the Study of Evolution in Action proposals
December 13, 2010
The BEACON Center for the Study of Evolution in Action is an NSF Science and Technology Center founded with the mission of illuminating and harnessing the power of evolution in action to advance science and technology and benefit society. Research at BEACON focuses on biological evolution, digital evolution, and evolutionary applications in engineering, uniting biologists who study natural evolutionary processes with computer scientists and engineers who are harnessing these processes to solve real-world problems.
BEACON is headquartered at Michigan State University, with partner institutions at the University of Texas at Austin, the University of Washington, North Carolina A&T State University, and the University of Idaho. At UW, the faculty include Ben Kerr, Herbert Sauro, Carl Bergstrom, Joe Felsenstein, Wenying Shou, and Billie Swalla.
Proposals for BEACON funding are due on January 10th. Please post any ideas for projects that involve evolution that we can discuss in this blog.