iGEM at UW
We have a world-champion (2011) undergraduate synthetic biology team that has competed in the iGEM competition every year since 2008.
Lab on a Strip: Developing a Novel Platform for Yeast Biosensors
The Pacific Ocean is home to a wide range of marine life, including the
food source of many filter-feeders, toxin-producing algae. When algal
blooms are ingested by shellfish, the toxins produced by the algae are
caught within shellfish tissue. Although these toxins are harmful to us,
they aren’t to the shellfish, giving collectors no immediate sign of
danger. Biotoxins are also just generally difficult to detect; contrary
to popular belief, algal blooms are not always the striking crimson of
“red tides.” Thus, blooms may not be discovered until after a poisoned
shellfish is found. The Washington State Department of Health and
commercial shellfish farmers conduct periodic surveys of local beaches
to catch contaminations early, but these methods are costly,
time-consuming, and not always effective. This can especially pose a
dilemma for individual shellfish hunters, who do not have the resources
to screen their shellfish for toxins. With current detection methods,
the crowds swarming to Seattle’s famous Pike Place Market and popular
raw oyster bars are constantly at risk.
We have developed a much cheaper diagnostic tool in which
genetically-modified baker’s yeast is grown on a paper device and is
able to produce an easy-to-read color output in the presence of a target
molecule. Imagine if you could simply dip a sheet of paper into your
bucket of shellfish, wait only (insert amount of time) and tell if your
products are safe to consume. The proof-of-concept systems we’ve
engineered detect the plant hormone auxin and the molecule theophylline.
However, we’ve implemented a number of techniques to ensure the
versatility of our systems thus, they can be easily modified and further
developed to test for a wide variety of other molecules.
2014High Throughput Selection of Stable Protein VariantsStabilizing proteins is an incredibly important and time consuming task in the field of protein engineering. Current methods require using intimate knowledge of the protein to hypothesize point mutations that could possibly improve stability. Extensive in vitro testing follows involving cloning the new construct into your model organism, expressing the construct, purifying your protein of interest, and producing a melting curve to verify if indeed your mutation did improve stability. In addition to being time intensive, this method is unreliably successful.
Our team has developed a generalizable, high throughput method to select for the increased expression and stability of engineered proteins, making them more amenable to large-scale production in Escherichia coli and other downstream applications. Our method involves the insertion of proteins into a Gal4-VP16 transactivator that binds a promoter directly upstream of a GFP gene. The protein of interest is inserted into the middle of this complex using polypeptide linkers allowing for the subsequent selection of mutants associated with higher GFP output. We hypothesize that more stable proteins and their complexes will not be degraded by the cell’s natural machinery which will allow the Gal4-VP16 construct to produce higher levels of GFP. Less stable proteins will be degraded through natural mechanisms and will not produce GFP. The difference between these two populations can be evaluated using Flow Cytometry or Fluorescence-Activated Cell Sorting.
Our method utilizes degrons to produce the desired range of GFP based off of the proteins relative stability while applying a destabilizing influence to the protein complex. The degron illuminates the differences between proteins of varying stabilities. This allows the system to operate with a higher clarity between stable and unstable protein variants. While the degron exaggerates differences between stabilities, it also can be used as a variable tool that can be adjusted to fit your protein of interest. By placing the degron in different positions along the protein complex, you can impose different destabilizing effects on the construct.
The end goal of our project is to create a system that can be used in today’s protein engineering laboratories. By using Fluorescence-Activated Cell Sorting of variants in a random mutagenesis library of our construct, we can sort out the highest GFP output variants which will correlate to the most stable variants. Through successive sorts the population will converge on a variant that improves stability of the protein complex. This revolutionary method is a generalizable alternative to current, labor intensive approaches for the selection of stable protein variants. Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics.
Red Light, Green Light
Recently, techniques have been developed that afford researchers the ability to control gene expression with light. To date, sensors that respond to red, green, and blue wavelengths of light have been reported. Light induced expression of genes has a number of advantages over chemical induction methods. For example, light induced expression is cheaper than chemical methods, can be used to finely tune expression levels through modulations of intensity, and can be rapidly removed -- a feature lacking in chemical induction systems. Furthermore, many light expression systems are reversible, depending on the wavelength of light used. Finally, the ability to control the expression levels of multiple genes of interest simultaneously could have far reaching implications for tuning biosynthetic pathways. The use of chemical inducers for this application would likely prove to be difficult and cost prohibitive, but multichromatic gene induction represents a potential solution to this problem. To this end, the 2013 University of Washington iGEM team chose to continue a project we began in 2012 that is aimed at the development and characterization of a set of tools that bring multichromatic gene expression into the realm of possibility for synthetic biologists.
In 2012, the University of Washington iGEM Team developed a freely available app for the Android operating system that affords spatiotemporal control over light wavelengths and intensities. When installed on an appropriate tablet (see E.Colight) the app could be useful for carrying out complex light induced expressions. In 2012, we were unable to fully characterize the app’s ability to control expression of light inducible genes. Thus, the 2013 iGEM Team’s goals include fully characterizing the app’s ability to control gene expression with light, and the development of a toolkit of light induced expression biobricks that are confirmed to work with the tablet app.
Biological systems must often be painstakingly tuned before they will efficiently produce drugs or biofuels, degrade chemicals, or perform other useful tasks. Our team implemented broadly applicable methods to optimize biological systems through directed evolution, light-regulated gene expression, and computer aided protein design. We characterized light-inducible protein expression systems for multichromatic tuning of biological pathways. To provide an inexpensive method for tuning gene expression with light, we developed a tablet application that is freely available. We also used computer-aided design to develop proteins that more effectively bind isotypes of the flu protein Hemagglutinin. Finally, we implemented a continuous culture device (turbidostat) in order to apply directed evolution to the metabolism of ethylene glycol in E. coli. We have termed the research conducted this year “Apptogenetics” as all projects utilize purpose-built computational applications for biological research.
Make it or Break it : Diesel Production and Gluten Destruction, the Synthetic Biology Way
Synthetic biology holds great promise regarding the production
of important compounds, and the degradation of harmful ones. This
summer, we harnessed the power of synthetic biology to meet the world’s
needs for fuel and medicine.
Make It: Diesel Production We constructed a strain of Escherichia coli
that produces a variety of alkanes, the main constituents of diesel
fuel, by introducing a pair of genes recently shown to convert fatty
acid synthesis intermediates into alkanes.
Break It: Gluten Destruction
We identified a protease with gluten-degradation potential, and then
reengineered it to have greatly increased gluten-degrading activity,
allowing for the breakdown of gluten in the digestive track when taken
in pill form.
To enable next-generation cloning of standard biological parts, we
built BioBrick vectors optimized for Gibson assembly and used them to
create the Magnetosome Toolkit: a set of 18 genes from an essential
operon in magnetotactic bacteria which we are characterizing to create
magnetic E. coli.
Antibiotics for the 21st Century
While vital to our quality of life, traditional antibiotics face the serious
problems of widespread bacterial resistance and destruction of natural gut
flora - problems which call for improved twenty-first century antibiotics.
Using synthetic biology tools, we designed, built, and tested two new
systems to fight infections by both broad types of bacteria - Gram-positive
and Gram-negative. Our first project targets Bacillus anthracis, the
Gram-positive pathogen that causes anthrax. We re-engineered an enzyme to
remove the pathogen's protective coating, rendering it defenseless against
the immune system. In our second project, we re-engineered and transplanted
a protein secretion system capable of combating Gram-negative bacteria into
E. coli. This system was designed to target Gram-negative pathogens in a
modular and controllable fashion. These two systems are the vanguard of a
new era of antibiotics using the power of nature harnessed with the tools of
Ideal Protein Purification
Recombinant, purified protein production is a decades-old technology
that has revolutionized research in biotechnology and medicine.
However, the traditional method of purified protein production is a
time-consuming and laborious procedure requiring expensive and
specialized equipment. Our project, the Idealized Protein Purification
(IPP) system, is an all-in-one protein expression and purification
platform built on BioBrick standards that will reduce costs, save time,
and simplify procedures associated with recombinant protein production.
The key to our IPP system is a novel combination of three subsystems:
expression, secretion and display. We use E. coli bacteria that
we have genetically modified to be a chassis for expressing your
favorite protein, secreting it to the media, then binding and displaying
the protein on the cell surface. At this point, collecting your
favorite purified protein is as simple as pelleting and
re-suspending a sufficient quantity of bacterial cells in an elution
buffer. The speed and simplicity of our IPP system exhibits the utility
of synthetic biology for developing new techniques that improve upon
novel abilities into eukaryotes has many potential applications. Our
project attempts to control transfer of genetic material across
phylogenetic domains. We attempt to direct the prokaryote Escherichia coli (domain Bacteria) to transfer DNA encoding potentially useful traits from to the yeast Saccharomyces cervisiae
(domain Fungi). The design utilizes standard engineering and synthetic
biology techniques to modularize this process, in order to enable usage
across varying organisms and conditions. To achieve control over our
system, bacteria transfer DNA via conjugation only if certain conditions
are met. In our design, E. coli transfers the genes to metabolize lactose in S. cerevisiae,
but only where lactose is prevalent, glucose is minimal, and yeast
proximity is sensed via a yeast-produced signaling molecule. It
therefore provides a means for conditional, not constitutive, gene
transfer between diverse organisms. Applications might include the
production of transgenic plants and animals, clinical gene delivery, and
interacting multiple-organism systems.