Integrated Human Practice
Cause every step counts, we were keen on ensuring that every idea is valid, and every step is right,
because every single step in scientific research makes a difference, a huge one actually. We managed
to do that by consulting professors from several universities all over Egypt, even if this means we
have to travel a very long distance to reach them, also consulting researchers in multiple research
institutes, and most importantly experts in the field of desalination.
Picking the chassis
It was not an easy task, and this took the longest time and effort, we looked up many previous iGEM projects, read a lot about
salinity stress and its effect on the cell generally, that was when we decided to work with plant as
our chassis. We went to ask all the doctors we knew in our faculty, we asked Dr Aya Ali, a plant
Biotechnology master student, she suggested that we should work with small plants, which are faster
to grow, and easier to handle in vitro, recommending Azolla, we went through intensive research to
view Azolla’s characteristics, its growth period and read literature about its genetic modification
feasibility, we further decided to ask other doctors about this candidate chassis. Dr Rehab Hafez,
Doctor in botany department was the first to be asked, she told us, “If you are willing to work on
plants, take into consideration the effort to genetically modify a plant” we were histating about
this fact, especially because most of the team are not so familiar with plants genetic modification.
Then we started again to search for other alternatives. We looked thoroughly in literature to find different microorganisms that are good candidates to be modified. On the other hand, we were putting in
mind that the chassis needs to have vacuolar compartment, as a huge part of our project greatly
depends on accumulating the salts in the vacuole. So, we turned to ask a specialist in stress genes
Dr Mohamed Hazman, researcher in AGERI (Agricultural Genetic Engineering Research Institute), we
explained the idea and our concerns to use plant chassis, he confirmed “Plants are hard to modify
their genetic content, take time to grow, and it will also be a problem to implement your idea into
industrial scale if you used them”, we considered this carefully. He recommended working on a wild
type that is capable of accumulating high concentrations of salts, i.e. microalgae “cyanobacteria” or
yeast, in order to confirm we went to ask professor of biochemistry Dr Ahmed Osman from Ain Shams
University about the choices we had, he said, “If you are willing to integrate the system, how you
are willing to deal with the odour and metabolites produced by microalga, you have to consider this
first”.
That made us recheck for the produced metabolites and again re-think about our choice. After
intensive research and brainstorming sessions, we decided to work with Debaryomyces hansenii as our
chassis and proof our concept first in E.coli since it is easier to be modified and much more
characterized.
The issue was how can we get the strains, can we isolate it? Or buy it from Egyptian Microbial banks
such as Mercn? we decided to do both, collect samples from the Mediterenean Sea and buy it in case of
isolation did not work out. We searched for Egyptian professors who had isolated Debaryomyces sp.
before, in the faculty of veterinary medicine in our University, we arranged a meeting with
Professor Salwa Aly, a professor of food hygiene department. We knew about her as she was one of the
authors of a paper published about yeast isolation from karish cheese, we asked “How did you
identify your isolates?”. She answered, “First by biochemical tests using API 20 c AUX, after that
molecular test using ITS”, and she recommended some textbooks and protocols for us. We wanted to confirm
more about the process of isolation, we met Dr Ahmed Osman, this time we went 227 km away from
our University, at the Egypt Japan University of Science and Technology in Alexandria Governorate,
we managed to arrange a meeting with Dr Ahmed, we asked “what’s the most preferable way to identify
the type of microorganism? Is it okay to depend on the biochemical test?”, he answered “Neither the biochemical test nor the RFLP will be as accurate as the 18S”, he continued
explaining about RFLP, the probability of finding restriction sites giving the same band size is
not that rare. Also, regarding the biochemical tests; the substrate is very expensive and there’s
a probability to find different yeasts working on the same substrate. We deduced that the most
effective way to identify the yeast is the molecular way. In the process of searching for the
Egyptian professors whom we can get the Debaryomyces sp. from, we have found Dr Nageh Abo Dahab, a
professor of microbiology and ex-head of botany department in Assiut governorate 400 Km far from our
University. When we went there, we asked about the favorable media to subculture the strain, Dr
Nageh recommended to use the media type according to the type of work we want to perform on the
yeast. We performed PCR for ITS and sent it to be sequenced in korea after this very long way to
come up with the chassis.
Choosing appropriate parts
In every iGEM project, parts are the vital factor upon which the idea forms and succeeds, when we thought of the first approach, we knew that increasing the sodium and chloride ions in the cell will increase cytoplasmic toxicity, leading to a series of devastating mechanisms, and eventually cause cell rupture, to avoid that we asked Dr walid Abou El-Soaad, an associate professor of plant physiology specialized in plant stress, about the impact of increasing cell toxicity upon elevated salinity stress, he said “You must consider in the first place the Reactive Oxygen Species (ROS) production and try to integrate your system in a way to avoid the impact of these toxic molecules”, when we searched we found that: beside the role of osmoprotectants in keeping the cell integrity, it is efficient to defend the cell against ROS. This advice have made a great impact on the choice of our part that we targeted to eliminate cytolysis. We have chosen to work on Trehalose osmoprotectant, we discussed this with Patrick Diep our iGEM mentor who advised us to also work on osmoprotectants that aren’t naturally expressed in Debaryomyces hansenii, which we later chose to be Myo-inositol.
As for the second approach it was very ambiguous for us, we hadn’t worked on such approach during our educational years, we consulted Dr Abdo el Fiky,an associate professor of biophysics, who was outside the country at that time, but he was pleased to help, he recommended I-tasser to model the synthetic protein. And when we told our mentor Patrick he suggested starting by characterizing the metal binding proteins found in the registry, as the binding process isn’t specific for certain metals, it was a great idea, we searched for the metal binding proteins in the registry and chose to order most of them to be characterized.
Water
Water is our main concern, so, before taking any further steps, we wanted to view the current technologies upon which the RO (Reverse Osmosis) and Thermal distillation processes depend on. We went to Waterx Expo conference that is held every year in Egypt and asked about every detail in the process, energy consumption, cost, drawbacks and any recommendations for how we should integrate our system in a desalination plant. We discussed with different engineers and project managers of different companies as Veolia, IWTE, IETOS, ISS, AquaNile and WatchWater, most of them recommended the use of our method as a pre-treatment stage in desalination to decrease the amount of salts in water before those salts pass through the reverse osmosis membrane, on the other hand we had a voice call with Engineer Mohmed Fathi in “live from water organization” he suggested implementing the system as a pre-treatment stage and ensuring its easy operation as non-experts could work with it.
Finally, we sought advice hearing more from technical support, researchers and experts. We contacted Dr Amira, a researcher at EDRC (Egyptian Desalination Research Center), who set up a meeting with Dr Hossam Shawky, head of EDRC and a water desalination expert. During the meeting, we discussed our project in details and asked about why specifically RO is the most used technique in water desalination, he stated that “RO is used in about 70% of the total desalination processes we make in the plants, it is very efficient in removing sodium chloride, where the latter is the most important and most difficult to be removed.'' He also recommended that we first test our hypothesis on saline, followed by artificial sea water and finally we could use actual sea water. We also met with Dr Mostafa Abo El-Fadl, a professor of water chemistry and head of water quality unit in the central lab, and Dr Dalia Elsayed Abozaid, an economy specialist with whom we discussed the current desalination costs; she stated that “Desalination cost differs from place to place according to the need for this water, in Marsa Matrooh the cost of one cubic meter is 13 pounds while in Haliab and Shalatin it costs more around 15 pounds for one cubic meter, because there one drop of water makes a difference”. She said that Egypt is now converting to desalination because we are in scarce need for it, each year a new desalination plant is built all around Egypt to face the soon-to-happen water crisis, she showed her concern. In fact, this visit was more fruitful than we could ever imagine!
Our visits didn’t just stop at EDRC, we also went to NRC (the National Research Center), there we reached for Dr Enas Abou Taleb, a researcher of environmental and occupational medicine department, who listened to our idea, and we asked her about the possibility of using a genetically engineered yeast inside the water, She said “In my opinion, a genetically modified yeast wouldn’t make a problem as it's already present in the sea water and it will just perform its job which is capturing the salts”.
At the Canadian Institute in Cairo, we met Dr Shereen Zahran, a water management engineer. We discussed the integration of our system to the desalination systems already present in Egypt. She said “Your system can be added as a pre-treatment step to support the RO system and increase its lifetime as this system will decrease the amount NaCl passing through the membrane”.
Hardware design
At first we had no idea how we would separate the yeast from desalinated water after finishing its task. We decided to design the hydrocyclone an easy to be done system, and use it in the separation process.
