Whether it is improving or discovering, science is all about sharing knowledge. It is crucial for the iGEM UNILausanne team to participate to this contribution to science and support future iGEM teams to find everything that could ease their experiment on this competition. Hence you will find in the following lines all the parts this year's team contributed to.
FfIBP is an antifreeze protein (AFP) variant new to the iGEM competition - we added the gene coding for this protein to the registry, as a basic part. We also created new composite parts, BBa_K3782001 and BBa_K3782022, to express this gene: we integrated it in two distinct vectors, pET-17b and pCold-I, that can be used by future teams. We performed various assays on FfIBP, measuring its TH and proving its use in preventing frost damage on Arabidopsis thaliana. We were therefore able to clone, purify and test this protein successfully, making it ready to use by future iGEM teams.
We further characterized an already existing AFP variant, RiAFP, by purifying the protein and performing various assays on the solutions we obtained. These assays allowed us to measure the TH activity of the protein and demonstrate its effectiveness at preventing frost damage on Arabidopsis thaliana. We also created new composite parts, BBa_K3782009 and BBa_K3782023, by inserting the RiAFP gene in the pCold-I and pET-17b vectors. In the pCold-I vector, the gene was placed under the control of the cspA promoter. Placing RiAFP under the control of this promoter is also new to the iGEM registry.
We also created new composite parts, BBa_K3782010 and BBa_K3782024, containing the gene coding for DcAFP integrated in the pCold-I and pET-17b vectors. Once more, the gene was placed under the control of the cspA promoter in the pCold-I vector.
We are the first iGEM team to prove that we can produce tailocins, which are phage-derived bacteriocins that specifically kill a narrow range of bacterial strains. We successfully extracted two solutions of tailocins from our killer strain Pseudomonas syringae pv. aptata DSM50252. The tailocin solutions have been proven to kill efficiently our target strain, Pseudomonas syringae pv. syringae B301D, as shown in our overlay assays (see Results). Since we are the first iGEM team to work on tailocins, we offer to the next teams interested on tailocins extraction an efficient protocol. Furthermore, we have proven that killing our target pathogen, which catalyzes the formation of ice due to their production of ice nucleation proteins at low temperature and therefore damages the plants’ membrane, can reduce the freezing temperature of water significantly with FROZONE (see Proof of Concept).
For our project, we needed a device to characterize the AFPs in their ability to lower the freezing temperature of water and to prove that we can lower freezing temperatures of water drops contaminated with pathogen strains Pseudomonas syringae pv. syringae by using tailocins to kill them.
We developed FROZONE, a low-budget self-made device that is able to change the temperature precisely to be able to compare two treated water drops with each other in freezing and melting temperatures as well as measure thermal hysteresis of AFPs.
We therefore designed it to have a precise control of the temperature on a copper plate, on which we placed our drops of solution. This plate is located in a vacuum to prevent ambient water from crystallizing. An attached microscope allows us to visualize the freezing process.
The machine allows us to drop the temperature on the copper plate from 20 °C to -20 °C in less than 10 minutes.
To contribute to future iGEM teams, we are uploading a complete 3D plan of our machine and a step-by-step tutorial on how to make it on the Hardware page. This will allow future teams to streamline the assay portion of any project involving ice crystallization phenomenons.
To be able to precisely control the temperature in FROZONE we created a software called EDNA which allows us to adjust the temperature to a setpoint with a minimized fluctuation on our device. We are making our software accessible in Github, allowing future teams to control their very own version of FROZONE immediately.
We also performed an assay on Arabidopsis thaliana to test our AFP solutions. To do so, we immersed plants in our AFP solutions and in simple buffers, then froze the samples overnight. We then dyed the plant using Trypan blue that enters dead cells and therefore highlights damage in the plant. Finally, we compared the treated plant leaves protected by our AFPs to the untreated ones simply frozen in a buffer. To do so, we designed an image-based software, VISION. This software analyzes the leaves and detects and measures the blue areas - which correspond to the damaged regions - of the plants. It then generates the fraction of the damaged area to the total one. We are also making this software accessible to future teams, on Github, allowing them to analyze their own assays on plant damage.