When looking through the list of project abstracts for the iGEM 2018 Team competition, we saw that the RHIT team was also working towards a very similar goal. They were engineering bacteria to produce PETase and MHETase to breakdown PET and then engineering the bacteria to metabolize ethylene glycol (EG) by overexpressing fucO and aldA. They had also done a lot of interesting mathematical modeling for EG metabolism. Since we had taken very different approaches in trying to work towards a similar end result, we thought that it would be incredibly enriching and beneficial for both teams to connect and discuss our projects. We immediately reached out via email and scheduled a virtual meet-up.
During the meet-up, we ended up covering quite a lot of ground and talked for much longer than we had anticipated. We began by talking about our respective projects as well as the logic and rationale behind our differing project designs. While our project was focused on completely eliminating PET by completely metabolizing all the PET degradation products, the RHIT team intended to collect the leftover TPA to be used for re-polymerizing into new PET plastic, an interesting idea we had not thought about before.
Next, although we were both working on engineering strains of E. coli to metabolize EG, we were using completely different methodologies, which we discussed at great lengths. Our approach was more centered on genomic engineering. Since two key enzymes (fucO and aldA) are known to improve EG metabolism, we had used Lambda Red recombineering to replace the native genomic operons for these two genes with inducible promoters, allowing us to control overexpression of each enzyme using IPTG and aTC induction. In addition, we had used MAGE to target the ribosome-binding sites of key genes identified from our Flux Balance Analysis modelling with the hopes of optimizing EG metabolism at the translational level. In parallel, we were also using a mutagenesis and adaptive evolution approach to gradually evolve an EG-metabolizing strain of E. coli.
The RHIT team, however, used none of these approaches. Instead, they were planning on expressing the two key enzymes (fucO and aldA) from a high-copy plasmid. By increasing the copy number of these key enzymes, the E. coli would be overexpressing the starting genes of the EG metabolism pathway and increasing the flux through the pathway.
Afterwards, we then talked about the different types of modelling we had done. Using Flux Balance Analysis, we had modelled E. coli metabolism under restricted growth conditions with only EG (or EG metabolites) as sole carbon sources. This allowed us to identify key genes that would need to be up-regulate and down-regulated for optimal flux through the pathway.
On the other hand, the RHIT team had used more of a kinetic model to predict how feasible it was for the E. coli to grow off EG as a sole carbon source and estimated doubling times under those growth conditions. Although we initially had trouble understanding their model, we eventually were able to grasp the way they had set their model up to be as predictive as possible. We also found it incredibly encouraging that their model predicted a fairly reasonable doubling time for E. coli growing off of EG, which meant that using a synthetic biology approach to solving the problem of PET waste was feasible.
Lastly, we talked about obstacles that we had each been facing and potential ways we could help each other overcome the obstacles. One of our main obstacles was the procurement of suitable PET samples to use in our downstream enzymatic activity assays. Initially, our last resort was to use PET pellets or water bottles. Luckily, the RHIT team had managed to contact multiple plastic manufacturers in their region and had collected quite a large variety of PET samples to use, ranging from powdered PET, to high/low-crystallinity PET, to PET films, etc…They generously offered to let us use some of their samples in case we ever needed them as we progressed with our project.
One of the main obstacles that the RHIT team was facing was challenges with secretion and challenges with cloning. With regards to secretion, they were initially trying to use a pelB secretion tag but were unable to achieve efficient secretion of PETase and MHETase. Since we had actually spent quite a lot of time testing various secretion systems as part of our Objective #1, we advised for them to try the yebF secretion tag, which worked extremely well for us and was relatively simple to use since the secretion system is native to E. coli. With regards to cloning, the RHIT team seemed to be having trouble getting their plasmids to transfect successfully into competent cells. We completely understood their frustration and cloning difficulties, having struggled ourselves quite a bit. To help troubleshoot their issues, we suggested two alternative cloning approaches that seemed to work fairly well for us: (1) Gibson Assembly and (2) Ligation-Independent Cloning. Both of these methods had worked well for us in the path whenever we faced problems with traditional restriction enzyme and ligase cloining.
All in all, we had a great experience working with the RHIT team and getting to known their team members. We found our meet-up with them to be extremely intellectually-stimulating and fun. During the Giant Jamboree, we plan on finding time to meet them in-person and talking more about how our projects are going.
While looking through postings for Team Collaborations on the iGEM Website, we saw a listing from the NAWI Graz Team looking for “Acinetobacter baylyi ADP1 support”. Since our entire Objective #3 revolves around engineering A. baylyi ADP1 to metabolize TPA, we immediately reached out to the NAWI Graz team. We also planned to send samples of A. baylyi ADP1 to them so that they would have a lot more flexibility working with the strain for future project directions. We are currently finishing up the paperwork and regulatory procedures required to mail biological samples internationally.
Since our projects utilize A. baylyi in completely different ways, we decided to host a virtual meet-up to discuss our projects and learn more about their team. Since we had not yet interacted with any iGEM teams outside of the United States, we were especially excited to experience the “i” for “international” in “iGEM” for the first time by collaborating with a European team.
Despite the six hour time difference that made scheduling a virtual meet-up a bit challenging, we were able to find a time for both our teams to talk. We began with a quick discussion about our respective projects. We learned about the innovative approaches that the NAWI Graz team was taking in order to produce palm oil using microorganisms. It turned out that the reason they were interested in using A. baylyi ADP1 was because A. baylyi ADP1 has some interesting metabolic pathways that could potentially help them optimize their production of palm oil. We found it incredibly cool that even though both of us were interested in utilizing the unique metabolic pathways found in A. baylyi ADP1, we were utilizing the same organism is wildly different ways. This really showed us the promise that synthetic biology holds for solving real world problems by creatively taking advantage and modifying biological systems already found in nature.
We also spent quite a bit of time talking about how each of our teams were structured. We were especially interested in hearing about how the NAWI Graz team had members specialized for web design, outreach, fundraising, etc… Since every student member of the Yale iGEM team conducts research, we do not have specialized team members working on specific aspects of the iGEM competition. We discussed with the NAWI Graz team the benefits and drawbacks of our differing team structures and were inspired to potentially try out their team structure in the future (i.e. having specialized team members for various aspects of the iGEM competition other than lab research). This would help take the load off the rest of our team so that they could focus on the lab research components without worrying about everything else.
Lastly, we talked a bit about how our teams were funded. Although both of our teams are heavily supported by our respective institutions, we both discussed the challenges of raising enough money to register for the competition, travelling to the Giant Jamboree, registering for the Giant Jamboree, and also covering research costs for the summer. We were especially impressed by the NAWI Graz team’s efforts in reaching out to companies about getting corporate sponsors.
Overall, we really enjoyed finally meeting a team outside the United States to see how iGEM is truly a competition that brings together students from all around the world. After meeting, the NAWI Graz team was kind enough to add us to their WhatsApp group message full of European iGEM teams who are attending the Giant Jamboree and are coordinating a meet-up. We look forward to meeting them all in-person and getting to know them more at the Giant Jamboree.