Better Together!
Meet our Partners in Delft! Early on in the competition we got in contact with the iGEM team TU Delft and their project PHOCUS. Due to the great similarity in our project ideas, we were able to exchange knowledge and establish a fruitful partnership. Read all about it below!
Meet the iGEM team TU Delft!
On May 29, a message from the iGEM team TU Delft reached us asking for a virtual meeting to discuss a potential collaboration between our teams. We were really excited about their interest in our project and met them for the first time on June 12. During this meeting we learned about their project idea PHOCUS and just how similar it was to ours
Our first video call with TU Delft! You can see Iris (Science manager), Gabriella (Outreach manager), Stella (Social Media Assistant), Oona (Team Leader), Katja (Second Wet Lab Manager) and Kathi (Social Media Manager).
What is PHOCUS?
PHOCUS presents a novel and safe approach to the locust plague in the horn of Africa, the Arabian peninsula and South Asia. Swarms of these locusts devastate croplands and threaten food security in the regions. PHOCUS is a biopesticide based on genetically engineered T7 bacteriophages that are ingested by the locusts. In the intestine of the locust, the phages infect the microbiome and incite the production of toxins. This kills the locust from within and reduces its harmful spreading in the regions. Find out all about it in their Wiki !
At the time of this first meeting, both our teams had decided independently on using an E.coli phage for our laboratory experiments as the work with E.coli was very well established in both our facilities. In the following days, we were debating within our team whether to use the E.coli phage Lambda or the bacteriophage T7. Finally, TU Delft’s decision to use the bacteriophage T7 was a factor that guided us in our decision towards T7. Despite the different desired outcome and the technical details of how we wanted to achieve it, our project ideas were so similar in what we wanted to achieve in the lab that we thought it would be interesting to use the same bacteriophage to increase comparability and to be able to exchange protocols.
In our discussion we also talked about how to select our recombineered phages once they were created. Our team already had designed primer sets that not only flanked the insert but we also had a pair of primers of which one partner flanked the insert and the other one was within the insert. This is important to verify the correct position of the insert in the phage genome. TU Delft considered this a good idea and subsequently took this into account for their project.
Of course, as it is necessary when talking about GMOs, we also discussed safety aspects of our projects. An approach like ours, where the phage is not able to replicate autonomously was not the right method for TU Delft as they need their phages to replicate within the host. We brainstormed a few ideas in one of our meetings some of which TU Delft elaborated then further for their project.
A summary of the most important differences and similarities can be seen in the table below:
| Differences & Similarities
| Phangel
| PHOCUS
|
| Recombination System
| Lambda Red
| Lambda Red
|
| Wildtype Bacteriophage
| T7
| T7
|
| Initial State of the Phage
| Linear DNA
| Complete Phage
|
| Desired Product
| Human Plasma Gelsolin
| Locust toxic molecules + RNA
|
| Application in which organism
| Human
| Locust
|
| Recombination Plasmid
| Self-Assembled by Golden-Gate Cloning
| pKD46
|
| Promoter of gene
| T7 Lac
| Natural promoter
|
| Selection Marker
| Kanamycin Antibiotic Resistance
| CRISPR/Cas9 system targeting the wild-type T7 phage
|
| Safety Mechanism
| Incomplete Phage Assembly
| none
|
Table: Differences and similarities in the iGEM projects PHANGEL and PHOCUS.
Because TU Delft used the complete recombination plasmid pKD46 and did not need to perform all the Golden Gate Cloning steps beforehand, they were able to start their phage recombination experiments earlier than us. At this stage, it was not clear if we would have enough time to refine our recombination protocols. Luckily, our partner stepped in and provided us with troubleshooting experiences from their recombination experiments. They shared with us the steps that they struggled with most and how they tried to overcome them in order to save us some valuable time.
Check out one of our plates from the plaque assay!
To gain more insight into the biology of our engineered T7 bacteriophages, we decided to conduct some shared experiments to characterize them once they were produced. Unfortunately, we both were unable to have our engineered phages available in time, so we decided to characterize the wildtype by performing the assays on it. We developed protocols for two different experiments which we then exchanged.
Our team developed an infection dose assay to determine the minimal dose of phages needed to clear a liquid bacterial culture and through this the infectivity of the examined phage. It would have been interesting for us to determine the time our engineered phage, whose major capsid protein is relocated onto a separate plasmid in the producer cells, needs to clear the liquid culture and see if its infectivity was impaired by this relocation. Team TU Delft developed a plaque assay to determine the diffusion speed and virulence of our bacteriophages. The experiments on the wildtype also showed some interesting results which can be seen below. By performing these assays on our wild type, we could gather valuable experience in how to perform these assays. Although we could not perform them with our engineered phages we did some important preparatory work which might be useful if the project would be taken to the next step.
Beyond the scientific insights, this collaboration was very valuable for us as we had the chance to develop a laboratory protocol completely on our own and have it tested by another team! As you can imagine, this did not go down completely trouble-free. On our first try of the infection dose assay, for example, we realised that we had made an error in calculation the concentrations so nothing happened in our assay. Luckily, TU Delft had not conducted the assay yet so we could send the correct dilutions in time.
Throughout the whole collaboration, we also discussed several times if we could also establish a collaboration between our Dry Labs. However, as the envisaged real life applications of our projects are vastly different, different factors are important for the extension of our projects in the Dry Lab. We did exchange about a few theoretical aspects of the interaction between E.coli and bacteriophage T7 but no shared project was developed.
To actually meet the people we were having so many online conversations with, we were also discussing a real life meeting. We would have loved to be able to visit our partners and the beautiful town of Delft. However, as so often this year, Covid-19 upset our plans. Even as the travel restrictions were already alleviated and the infection numbers were slowly decreasing in mid-summer, both our teams did not feel comfortable with such a trip just for the pleasure of seeing each other and we decided it would be most responsible to continue relying on online meetings.
Results of the Experiments
In an infection dose assay and and in a plaque assay, we tested the interaction of our wildtype T7 phages with E.coli.
You can find both detailed protocols here. If your browser does not support embedded PDFs you can open the protocols seperately: Plaque Assay_BOKU-Vienna & Infection Dose Assay_TUDelft
For the plaque assay proposed by team TU Delft, the plaque diameter was repeatedly measured to monitor the growth of plaques over time. Several dilutions of a phage stock solution were used.
Results of the plaque assay of team TU Delft.
Results of the plaque assay of team BOKU-Vienna.
To simplify the interpretation of the data, the growth rates were treated as constant over time. Further, independence of phage number with respect to growth rate at the beginning was assumed. Therefore, the growth rates can be averaged.
The results showed a growth rate of 0.37 mm/h for Team Delft and 0.48 mm/h for us, respectively. Since we used the same phage, the result shows a lack of robustness and validity. This was expected since both teams are not experienced with the development of protocols and the experiments were only conducted once. However, more data would be needed for further protocol development.
As mentioned above, our team developed an infection dose assay. It determined the rough amount of phages which are needed to clear a growing batch process of the susceptible organism. This result can be useful for later upscaling in process development when larger amounts of the phage are needed. As an optimal inoculation dose, we determined a ratio of 1.3 PFU/bacteria. Team Delft monitored only a slight decrease in bacterial growth. Further experiments and repetitions are needed to find the reason for this result.
To sum up, it was very visible to us how hard it is to develop universally applicable protocols. However, still we got new insights about the properties of our phages.
What’s left to say...
Over the course of four months of shared objectives, scientific exchange and mutual support, we both felt like our collaboration had grown into a partnership on September 30. We truly enjoyed collaborating with the iGEM team TU Delft over the course of these now (almost) five months. Their ideas and insights were very inspiring and we believe their advice shaped our project for the better. Thank you very much for this amazing time!
Dank U Zeer!
