This year’s UCSF iGEM team was a collaborative effort between several institutions. Because UCSF has no undergraduate program they partnered up with: my high school, Abraham Lincoln, because we had an intensive biotechnology program taught by George Cachianes and Julie Reis, and one student from Palo Alto High School and one from UC Berkeley.
Using iGEM as a guiding gramework we came to a conceptual focus: we wanted to address the significance of space within cells. As synthetic biologists, we frequently engineer biological systems by simply introducing novel functionalities. Rarely do we care about the myriad roles space can play on cellular behavior, and as such, our team sought to address the significance of spatial localization by directing biology through synthetic assemblies and organelles.
We take for a fact that “the processes that cells carry out are complex and many.” Such as: to harness energy, maintain homeostasis, or respond to environmental cues, cells must execute numerous ordered, multi-step and often interrelated pathways. As such, doesn’t it seem like a logistical nightmare to have the right enzymes, substrates and intermediates for all of these pathways freely diffusing around in the cytoplasm? This task only becomes more daunting as one moves beyond bacteria; complexity of eukaryotes.
Obviously cells have evolved to avoid this potential mess. Otherwise we wouldn’t be here. What’s more, they’ve done so with very simple solutions: spatial organization. By selectively localizing and organizing particular cellular components in different regions, messages can be quickly and accurately sent, molecules can be correctly synthesized, and results are achieved. This spatial localization can be achieved through two different mechanisms: the formation of protein complexes (organize/regulate) and compartments (physically isolate interior environment from cytoplasm).
Protein Complexes can specifically control the wiring of a cellular circuit by regulating both its composition and its connectivity. This simple approach can create powerful results. Frequently, proteins are organized upon scaffold proteins, where multiple, specific docking sites specifically recruit interacting partners. This prevents cross-talk while also executing multi-step tasks efficiently.
Compartments: distinct, membrane-bound compartments within a cell also offer one ultra-powerful advantage: the “business” of the interior does not interfere with “business” of the cytoplasm (vice-versa). Unique environments can thus be created within (pH, unique concentrations). Higher local concentrations can be easily achieved by the benefit of a reduced volume. Moreover, controlling what’s inside and outside can create specific protein-protein interactions. As a result, cells can power specific circuits, functioning in specific environments. And, the compartmental membrane can either insulate this circuit from the outside environment, or insulate the outside environment from this new circuit.
The power of space within the cell has not yet been harnessed by synthetic biology. As such, our iGEM project approached the manipulation of spatial organization through two distinct projects.
First, by treating the scaffold as a “molecular breadboard,” we sought to rewire a kinase signaling pathway to build new cellular circuits.
Our second, and more ambitious project was to create a stable membrane compartment within a eukaryote: we wanted to create a new organelle. We christened it the “Synthesome.”
After this is accomplished, the options are nearly limitless for its function! Two very relevant applications are the use of the new organelle as a Drug or Biofuel factory.
Project I: Using a protein Scaffold to Rewire a MAP kinase signaling pathway
In our first project, we treated the scaffold protein as a molecular breadboard. We chose to specifically work with the scaffold protein Ste5, which organizes the MAP kinases in the yeast mating pathway. Because this kinase cascade is conserved in all eukaryotes, it is closely examined and well-characterized. Responding to pheromone outside of the cell, yeast transduce an external cue through this cascade to ultimately reach the nucleus, where transcriptional changes cause a mating response. Ste5 is crucial in transmitting this signal–without the organizational help of the scaffold, the MAP3K, and MAP2K, and MAPK are unable to effectively communicate. As a result, the mating response cannot be produced.
In our project, we plugged in an additional interaction site to the scaffold by fusing a synthetic leucine zipper domain to Ste5. This serves as an additional docking site for any protein that contains the complimentary half of the leucine zipper. Using this strategy, we recruited negative effectors that would repress the activity of the kinase cascade, and thus, modify the pathway output. We were able to measure the output of the pathway with a mating pathway induced GFP reporter.
Since we wanted to be able to effect a wide variety of dynamic changes, we created a toolkit of three different negative effectors, all of which were borrowed from bacteria. YopH reversibly inhibits the MAPK by cleaving the phosphate group on the Tyrosine residue that determines the MAPK activity. YopJ works irreversibly on the MAPKK by acetylation. Our third effector, OspF, is also an irreversible inhibitor, but it cleaves both the phosphate and the R-group from the MAPK regulation site. Because these three effectors vary in substrate, catalytic activity, and dynamics of inhibition, they form a very useful toolbox for engineering the mating pathway.
Experimentally, we wanted not only to compare the range of effects through the effector toolbox, but also to examine the significance of recruitment of each effector to the scaffold. So, for each experiment, we created three variants: one with No Effector, one with Effector Recruited to the scaffold, and one with an Effector Unable to be Recruited to the scaffold. We hypothesized that, compared to the full output of the undisturbed pathway, we should see a strong repression without actual recruitment to the scaffold.
**After personally cloning the YopJ and YopH effector constructs, I generated the YopH and YopJ graphs using FlowJo after running FACs experiments.**
After toiling in the lab over our constructs, we were able to experiment with all the conditions we desired. Moreover, a remarkable thing happened!
The results are clear!
AND, they closely followed my predictions!
Across the board, the recruited effectors drastically decreased the mating output of the pathway (as measured by GFP fluorescence by FACs). These same effector proteins, when unable to bind to the scaffold, had significantly weaker effects. Additionally, the results showed that the irreversible negative effectors (YopJ and OspF) showed stronger repressive potency.
Our final step in this project was to build in feedback loop to the circuit. All of the previous experiments were performed with constitutive expression.
In order to build a negative feedback loop, we placed the expression of the negative effectors under the control of a mating-induced promotor. As such, activation of the mating pathway induced expression of an effector, until its expression proceeds to shut off the pathway. This will create dynamic adaptive behavior after continuous stimulation with the pheromone (alpha-factor).
The results confirmed this behavior–as you can see, after 40 minutes of pathway activation, the expression of GFP comes to a halt.
Project 2: Building a New Organelle for Synthetic Biology
The existence of different organelles bound by distinct membranes necessitates a system by which the cell can distinguish different membrane locations.
One of the most well-understood methods of tagging such membranes is the phosphoinositide system.
Phosphoinositides are lipids that make up a small percentage of membranes. They consist of a hydrophobic tail and an inositol sugar head that can be phosphorlyated in three positions.
In yeast, there are no insotides tagged solely at the 5′ position.
Because of this, if we created this new tag in a specific location, we would theoretically obtain a modified vesicle to serve as a chassis for subsequent recruitment of other proteins, which through modular domains will be able to bind only to the desired phosphoinositide.
We have chosen the following strategy: express in yeast the higher eukaryote lipid phosphatase MTM, which catalyzes the removal of the 3-phosphate from PI[3,5]P2, to generate PIP in a specific location.
The endogenous system in yeast has a membrane associated mating-pathway receptor which binds a ligand (mating pheromone).
After ligand binding, the receptor-phermone complex is endocytosed. After endocytosis, an endogenous kinase phosphorylates the yet untagged phosphoinositide on the 3′ position.
As the early endosome is absorbed into the late endosome, a second endogenous kinase phosphorylates the 5′ position, resulting in a PI[3,5]P2 tagged endosome. This endosome is recognized by the vacuole, and is then targeted by its merger machinery for lysis.
In our strategy, human or worm MTM will be recruited to the membrane receptor of the mating pathway (Ste2). Pathway induction with mating pheromone (alpha-factor) results in endocytosis of the receptor (and therefore the recruited MTM).
Once in the late endosome, the MTM will convert PI[3,5]P2 into PIP, leading to Ste2-specific PIP-tagged endosomes.
These endosomes are expected to be stable, for they are unlikely to be recognized by the vacuole merging machinery.
Once we have created all of the constructs and yeast strains required, we have to confirm the creation of the synthetic organelle.
We plan to use two main methods:
The first is to use flourescence microscopy. The receptor that is endocytosed is fused to GFP, and a “Plekstrin Homology” (PH) domains specific for PIP is tagged with RFP. In the control strain, before induction, we should see a ring of green flourescence, representing the membrane associated receptors. After induction, there should be specks of green, indicating that the receptor has been endocytosed. Finally, we should see a green glow from the vacuole, as the endosomes are degraded. In the experimental strain, before induction, we should also see membrane associated receptor. After induction, we will also see endocytosis, but after some time has passed, we should be able to see GFP/RFP localization, in specks around the cell, indicating the existence of 5′ phosphoinositides lining the membrane of new organelles. Radioactivity techniques will be also used to detect the production of PIP.
We were able to modify the mating pathway receptor with GFP and tether the MTM lipid phosphatase. The receptor was still properly localized, functional, and able to undergo ligand-induced endocytosis. Furthermore, we could detect trafficking of the modified receptor to endosomes by microscopy.
This concludes the project that I worked on at UCSF for the 2007 iGEM competition