Team:Athens/Partnership
PARTNERSHIP
The Partnership part was written with mutual efforts of iGEM Athens & iGEM KU_ISTANBUL
Introduction
During this odd year, iGEMers around the world wanted to connect with each other more than ever, especially after the announcement that the Giant Jamboree will be held online. The need for a feeling of community, but also for collaboration partners pushed our team to look for other iGEM teams that work with light (electromagnetic radiation) and matter interactions. While searching through the database of the Maastricht Journal initiative, during early summer, we stumbled upon the work of iGEM KU Istanbul. We were initially drawn to their work because we found biological lasers an interesting topic, a bit relative to our project. We decide to contact them and the rest is history!
Our partnership was built upon different stages of our project development, from helping each other understand the problems we address, to assisting the design of our solutions and defining their future perspectives. We arranged a first get-to-know teleconference, where we introduced ourselves and our work, but also discussed how the pandemic had affected us, the mutual difficulties we were facing and the constant changes we were going through. After that first meeting, our team realized that the situation and the problems young students and scientists face are very similar. Besides the pandemic effects, the financial issues both of our teams experienced, brought us together and helped shape our partnership. The main characteristics that prevailed were mutual support, empathy and communication to help each other meet our goals.
At our following meetings, we presented our projects to each other in detail and exchanged valuable feedback. It was one of our first presentations for our team and we really appreciated the experience and the welcoming atmosphere. During those meetings, we discussed the extra needs each team had and iGEM KU Istanbul was in need of some parts from last year’s distribution kit. We gladly checked if we still had all those parts from iGEM Athens 2019 team and since we were not planning to use them, we mailed them those parts. In addition, the team of iGEM KU Istanbul was the first team to enthusiastically participate at our flyer translation on dyes.
After those meetings we found ourselves arranging a series of interviews, by suggesting to each other and helping contacting experts related to our fields, in order to get feedback from them. More specifically, the Istanbul team introduced us to Yilmaz Aslan, a Turkish former art teacher and artist, to talk about how the end product we are creating can be used by students. Full interview can be found here.
Similarly, we introduced the Istanbul team to Dr Mara Simopoulou, an Associate Professor of Physiology and Clinical Embryologist/Geneticist at Athens Medical School to help them understand the challenges of In Vitro Fertilisation (IVF) and how their laser could contribute to the characterisation of oocytes.
They also found a thesis that provided detailed information on the accidents related to that specific field in Turkey, a neighbor country to Greece. They also got out of their ways to help us translate parts of that thesis and to focus on what is directly related to our project, even during our not-so-rare after midnight working schedule. Finally our collaboration evolved with mutual help on wiki building and content proofreading. The feedback acquired was crucial for the presentation of our teams.
Project Collaboration
Our partnership could not be completed without collaborating to link our projects. Since both of our teams have not completed our projects due to the circumstances that prevailed due to the pandemic, our partnership focused on a potential future interaction and the design of future experiments.
It would be of significant importance for the MORPHÆ project that we are able to track the bacteria, as gliding motility has been linked to structural colour. Therefore, combining our work with the Living Laser was the way to achieve real time tracking! In order for this to happen, Flavobacteria cells need to produce reflectin in the periplasmic membrane as well as to express green fluorescent protein (GFP). After cells reach this state, the laser is pointed towards them, and can be tracked!
Genetic Engineering
To accomplish this, we firstly need to genetically engineer the flavobacteria to produce the above proteins in the desired cell compartment, but we would also need the hardware equipment from KU Istanbul team’s project.
For the genes design and insert, the following procedure is followed. The OPH signal peptide is designed to transfer the reflectin to the periplasmic membrane [1].
Additionally we used the promoter, RBS and terminator, (BBa_K3520007, BBa_K3520008 and BBa_K1172903) to ensure its successful translation at Flavobacterium species. The same procedure was followed for the GFP without the signal peptide, as it was not needed. Those genes are incorporated into the Himar transposon that is found in pHimarEm1 plasmid, using Type IIS assembly (level 2) standard. The plasmid containing transcriptional units (GFP and reflectin) are inserted with conjugation in the flavobacteria using the E. coli S17-1 as the donor of plasmid, following the same protocol as the one described in detail in the project description tab. After conjugation occurs, the two transcriptional units are transported into the Flavobacterium species genome.
Hardware
Below, the hardware of KU Istanbul that will be used to track the cells is described in detail.
Optical Setup - Configuration 1
The pump laser (Nd:YAG Pulsed Laser) with a pulse width of 5-8 ns which excites the gain medium molecules initially arrives at the optical parametric oscillator (OPO) where the wavelength of the laser is tuned in the visible spectrum by the help of a nonlinear beta barium borate (BBO) crystal. Power of the laser light coming out of the OPO is adjusted by suitable filters. Power adjusted laser light arrives at the dichroic filter where only the light of wavelength above a certain limit can pass through. If the right dichroic filter is chosen, only the pump laser will be reflected while the emission from the sample will pass through. In this way, the analysis of the emission from the sample will be easier since it is separated from the pump laser on the dichroic filter. Pump laser reflected by the dichroic filter will be reflected by the mirror and focused onto the sample by the microscope objective. Emission from the sample will be collected by the microscope objective, and reflected through the dichroic filter by a mirror. Since the chosen dichroic filter will allow the emission to pass through, it will arrive at the beam splitter without any change. The beam splitter splits the coming light into two pieces, half of the light passes through it, the other half is refracted by it. Thus, the emitted light can reach both the CCD camera and spectrograph. Sample image can be taken by the CCD camera with 0.03 nm resolution, and the emitted light can be analyzed by the spectrograph.
Pump laser: 10 Hz Quanta-Ray INDI pulsed neodymium-doped yttrium aluminum garnet (Nd: YAG) laser with a pulse width of 5-8 ns.
OPO: BasiScan optical parametric oscillator (OPO). (SpectraPhysics)
Spectrograph: Andor Shamrock 500i Spectrograph mounted with Andor Newton Cooled Spectroscopy Camera with 0.03 nm resolution.
Optical Setup - Configuration 2
The second configuration of our optical setup includes an incubator built in-house. This configuration is the exact same setup of the first configuration, the only difference is the incubator. In this configuration, the pump laser arriving at the microscope objective is passing through the transparent incubator wall and excites the gain medium present in the sample. The emission from the sample is being collected in the same manner, the emitted light is passing through the transparent wall of the incubator and collected by the objective.
After all that communication, mutual help and support as well as the bond created between the two teams, we promised to meet in person after the pandemic! Until then, the least we could do to celebrate our shared experience was to stream the Giant Jamboree together and enjoy the event connected!
"Butterfly wing" by Fernan Federici is licensed under CC BY-NC-SA 2.0
