Team:Greece United/Notebook


29/11 - 05/12

November 2020

After long research, reading papers on different subjects and various projects from the past years we concluded that our project would concern Osteoarthritis and in particular we would propose a new approach for the treatment of this musculoskeletal disease. You can find more about our final idea on the following link.

We made our final decision about our project on December 3rd.

06/12 - 12/12

December 2020

After we concluded on the project we studied more specific things about Osteoarthritis in pathophysiological and molecular level so that we had a clearer idea on what is happening in a joint when a patient suffers from Osteoarthritis. We tried to answer the following questions: How it begins? What causes it? Which molecules and cell types play an important role in an Osteoarthritic joint? Which are the existing therapies for this disease? What should happen in order to stop the progression of the disease.

Trying to answer these questions we had the need as a team to have a meeting with someone who has experience with this disease and is a professional Orthopaedic Surgeon. For that purpose, we prepared a presentation about our team, the competition and our idea as it was in the beginning and contacted Dr. Georgios I. Drosos (Orthopaedic Surgeon and a Chairman at the University Department of Orthopaedic Surgery and a Professor of Orthopaedics in the School of Medicine, Democritus University of Thrace). Thankfully Dr. Drosos was very interested in our project which led to his recruitment in our team on 10/12 as a PI to help us throughout the duration of the competition.

First iGEM Stuff Call : December 11th

Also, on the 11th of December we held our first igem stuff call to have a session discussing all the things concerning the iGEM competition. We held a weekly “iGEM stuff” call every week after that to discuss all terms and conditions of the competition, the deadlines, the medal criteria, the awards, the registry and the safety form, in order to make sure that the whole team has a clear idea of what we need to do in the competition in order to succeed.

13/12 - 16/01

As it is expected every team in a competition of these standards as the iGEM, needs a financial support, so in this week we had to compose our project proposal containing a meticulous description of the competition, of all the members of the team, including the Principal Investigators, a very descriptive analysis of our project concerning both the wet lab and the dry lab and last but not least the budget that we were expecting to need for the implementation of our project.

At the same time, a pitching presentation was prepared containing a short description of the competition, our team, our project and our needs in order to present when holding a meeting with a candidate sponsor. On 16/01 the first version of our proposal and pitching presentation was formed.

Our wet lab team was focused on understanding every aspect of the pathophysiology of Osteoarthritis at a molecular level. We started our first efforts to select which cell type is the most suitable for our approach and which factors should be expressed on our genetic circuit forming a primitive design of our experiments and writing it down on the project proposal. (for more information on what was the thinking process in order to conclude on the experiments we run you can click on the following link.

17/01 - 30/01

January 2021

On 17/12 we contacted the first companies which we were hoping to be our sponsors while the project proposal was being daily updated with additions of every aspect.

The wet lab team was perfecting the text on the project proposal concerning the Osteoarthritis and the Wet Lab experiments. We also formed a list containing the most promising factors to be included in our genetic circuit, where we added new things as soon as we learned about them. We aimed to find 3-4 factors in total, which would affect the anabolic and the catabolic pathways in the articular cartilage and the pain that the patient is experiencing during Osteoarthritis.

31/01 - 22/02

We took a break for the semester exams of our departments.

23/02 - 28/02

February 2021

The Wet team Lab started analyzing the sequences of our genetic circuit and choosing the vectors in which our genetic would be inserted. We held our call on 25/02 to discuss our ideas.

We started studying in order to choose whether the transfection of the cells in our approach would be transient or stable. We considered all the advantages and disadvantages of each method both for our approach as a treatment and the feasibility of it to implement it in the Lab. We held our call on 28/02 to discuss our ideas.

01/03 - 07/03

March 2021

The Wet Lab of our team started reconsidering the genetic circuit and exploring other alternatives based on RNA circuits. We studied papers considering the RNA circuits and tried to design an RNA circuit that would be suitable for our approach. We held a meeting on 7/03 to discuss our new findings.

08/03 - 14/03

The wet lab studied ways for measuring the factors expressed by the genetic circuit and design of the experiments. We researched and studied in order to find the protocols and kits most suitable for our approach. We held a meeting to discuss our new findings on 14/03.

15/03 - 21/03

Another concern that we had to resolve was what experiments we should run in order to be sure that our circuit produced the expected factors and would eventually have effects in an osteoarthritic joint. We tried to find a way to simulate the Osteoarthritic environment in vitro or ex vivo while excluding the possibility of running in vivo experiments due to the restrictions of both time and the competition. We held our meeting on 21/03 to talk about the new things that came up.

In parallel, our team started thinking and researching what needed to be done in order to open a crowdfunding page.

22/03 - 28/03

Our Wet Lab team had done a lot of research. We were at a very good level of knowledge regarding Osteoarthritis at all levels, and specifically at a molecular level. But, we had many alternative approaches for our project, each one accompanied by different protocols. Therefore, we needed the opinion of an experienced one, working on Osteoarthritis at a molecular level to help us decide which were the most promising and at the same time most feasible of our alternatives.

For that purpose, this week, we worked on preparing a document which meticulously described our idea and the steps that we were planning to make in the laboratory (experiments and protocols). We contacted, the one to later become one of our Principal Investigator, Mrs. Maria Margy Koffa. Mrs. Koffa is a professor on the department of Molecular Biology & Genetics, head of the Cellular Biology Laboratory and has had previous experience on Osteoarthritis.

Also, our team concluded on what needed to be done in order to open a crowdfunding page and from that point on (25/03) we started designing our crowdfunding page.

While posting various scientific related posts on our social media throughout this whole time, our project reveal started on 27/03. We prepared a total of 7 posts dedicated to our project with many hints and riddles for our followers to find our project idea.

29/03 - 04/04

Our team finished its project reveal on 02/04. By that time, we had already designed our crowdfunding page and eventually started its promotion on 04/04.

05/04 - 25/04

April 2021

As the deadline for the regular registration team fee was closing in, the whole team focused more on collecting the necessary amount of money for our registration to the competition. The promotion of our crowdfunding page continued while our team daily contacted many companies to find as many sponsors as possible.

During that period our Wet Lab, while facing some difficulties with the genetic circuit, decided to consider other possibilities. We examined the use of an RNA circuit instead of the DNA genetic circuit. We studied about RNA circuits in general and we tried to design an RNA circuit suitable for our approach.

Another aspect that we had to consider and conclude, was which factors would be expressed by our Circuit. So, we expanded our range of factors to small interfering and micro RNA molecules as well, as they seemed to have many potential as therapeutic molecules.

26/04 - 02/05

Based on our research, an idea came up to surface. We thought of using exosomes as a means of transport for the factors that would be produced by the modified cells. So, we studied about the use of exosomes as a therapeutic tool and learned about the fundamental mechanisms on how exosomes are being produced and how they can be modified. We found out that various Guiding tags could be fused to the membranes of exosomes so that they could specifically target certain cell types.

03/05 - 09/05

May 2021

Concerning the use of exosomes, we found out that a specific guiding tag has been used on the membrane of exosomes to guide them specifically on Chondrocytes when excreted which caught our attention as this was the cell type that we wanted to target.

As the time was passing, we needed to conclude on a certain approach. But we were unable at that point. Therefore, we contacted Mr. George Skretas, a successful synthetic Biologist in order to draw some help. For the purpose of our meeting, we prepared a presentation which included all of our thoughts and alternatives for our project to discuss them with Mr. Skretas and understand which were the most promising and which were the easiest to implement.

10/05 - 16/05

After our preparation for the meeting, we had a call with Mr. Skretas on 11/05. He helped us understand that the use of RNA circuits was a little difficult, mainly because of the short life of RNA molecules. Therefore, our initial DNA circuit was still a part of our approach. After the discussion about the exosomes, we concluded that this was indeed a viable approach and we had high hopes of sticking to this idea, while he also encouraged us to work with miRNAs as well.

Mr. Skretas also suggested contacting a fellow scientist Mr.Garinis, who was running a project at that time using exosomes to get some more feedback on the use of exosomes.

He later helped us to contact the designers of the genetic circuit that we were studying, Mr. Roman Jerala, in order to request the plasmids and their restriction maps.

On 13/05 we finally reached Mrs. Maria Margy Koffa and held our meeting. We presented to her our presentation concerning the iGEM competition, our team and our project. We had a discussion to gain some feedback, as she used to work on Osteoarthritis, then we discussed about her recruitment as a Principal Investigator and the possibility of running our wet lab experiments in her Laboratory (Cellular Biology Laboratory, Democritus University of Thrace, Dpt. of Molecular Biology & Genetics) both of which ended with a positive response.

17/05 - 23/05

Finally, after many indications following our research, we concluded on using miRNA-140 as the factor expressed by our genetic circuit.

We also contacted Mr. Garinis after the suggestion of Mr. Skretas, to hold a meeting concerning the use of exosomes. He directed us to his PhD candidate and member of his lab George Niotis, who was very willing to help. We finally had our meeting on 21/05, during which he explained to us many things about the exosomes, the ways in which we can use them and the protocols that we can use in order to work with them.

This, led to our decision to include the exosomes in our project as a means of transport of the factor (miRNA-140) produced by our genetic circuit.

24/05 - 30/05

On 24/05 with the help of Mr.Skretas, we contacted Mr. Roman Jerala, the original designer of the Genetic Circuit that we were going to use, requesting to send us the plasmids containing the Genetic Circuit and their restriction maps so that we could work on the design of the plasmids.

On 27/05 we visited for the first time the laboratory in which we would run our experiments. We met the rest of the members working in this lab and got a little familiar with the facilities of the laboratory. We also programmed a meeting with our PI Maria Margy Koffa, to have a little more specific discussion about the protocols we were going to use and the consumables we had to order. So, after that the Wet Lab team began to study more specifically about the protocols of the experiments we were planning to run.

The plasmids were sent on 27/05 from the laboratory of Mr. Jerala but unfortunately the restriction maps were not found so that they could be shared with us.

31/05 - 06/06

On 31/05 we applied for the Promega iGEM Grant Sponsorship.

On the first day of June (01/06) we held our meeting with PI Mrs. Koffa. She gave us an indication of how difficult it is to transfect a single cell with 5 different plasmids (the genetic circuit that we had in our minds) and also discussed how difficult it is to handle Mesenchymal Stem Cells in the laboratory. She therefore prompted us to reconsider our design, to determine more carefully which cell type we were going to use and form a very meticulous list of all the protocols that needed to be run about each and every step of our project.

We concluded that we were only going to use one plasmid, testing only the part of the Effector. We also decided to use HEK293T cells instead of Mesenchymal Stem Cells in our experiments, as they are easier to handle in the laboratory.

On 03/06 we acquired Geneious Prime License and we started designing our plasmid.

07/06 - 13/06

June 2021

The research was continued on 10 different steps considering our experiments. In particular the 10 steps were:

 -   Plasmid design
 -   Plasmid amplification
 -   Digestion of plasmid and insert & Ligation
 -   HEK293T cultivation
 -   HEK293T transfection
 -   Transfected cells selection
 -   Exosomes Isolation
 -   miRNA quantification
 -   Chondrocytes from Osteoarthritic tissue cultivation
 -   Western Blot for quantification of miRNA effects on chondrocytes

On 10/06 we successfully registered with Integrated DNA Technologies as part of the iGEM 2021 Competition.

We planned on ordering our plasmid backbone and the sequence of our insert from IDT as it was less than 3000 bp long. The complexity of our insert though was very high so as to be ordered from IDT, so we had to make some modifications. Our team was constantly working in order to overcome this problem.

On 13/06 we applied for the Beckman Bioreactor Quiz, Training registration, Reagents tool box and Lab bench devices tool box.

14/06 - 20/06

We worked on writing our preliminary safety form during the whole week.

We also focused on our plasmid design, searching for the sequence of every part of our insert, while also looking for the appropriate plasmid backbone

The protocols were pretty well documented, but we needed to specify certain things in order to move on with an order for the needed consumables. So, we were ready to have a meeting with our PI Koffa to verify that everything was well planned in order to proceed with our orders.

21/06 - 27/06

All of our experiments and protocols were determined very precisely, except for our plasmid design. Our team was ready to hold a meeting with our PI Koffa while we were constantly working on designing our plasmid (both the plasmid backbone and our insert) on Geneious.

We also prepared our application for the Impact Grant.

28/06 - 04/07

Our Wet Lab team held a meeting with Mrs. Koffa on 28/07 to discuss the protocols and the orders. We verified that everything was well planned although it was stressed that we were running out of time. The work for our plasmid design continued.

We applied for the application for the Impact Grant on 30/07.

We designed our plasmid with all the sequences and the appropriate prefixes and suffixes and we held another meeting with our PI on 02/07 to verify that the plasmid was well designed.

05/07 - 11/07

July 2021

Our first effort was made to find a HEK293T cell line from a nearby laboratory on 05/07. On 06/07 we contacted Addgene for a discount as we were prepared to order our plasmid backbone. Some modifications were made on our plasmid design and we changed the restriction sites in order to use some restriction enzymes that already existed in the Laboratory we would work in. On 09/07 we were offered a discount from Addgene to order our plasmid backbone.

Our team also prepared for an iGEM oriented webinar for the Anatolia College CTY

12/07 - 18/07

On 13/07 we held our webinar on the CTY college.

We were planning to order our plasmid backbone from Addgene and have our insert ordered from IDT as a gene fragment since it was less than 3000bp. The gene fragment that we wanted to order was characterized as a high complexity one, which was a barrier for ordering it from IDT. We struggled to overcome this and we did this through several codon optimizations, using various tools.

We also prepared for the iGEM Greece Meet-up which would be held on 24-25 of July.

19/07 - 25/07

The plasmid backbone was successfully ordered from Addgene on 20/07.

More preparations needed to be performed for the iGEM Greece Meet-up, which eventually was held on 24-25/07 where our team presented our project idea and the troubles we had faced during our course in the competition until this point.

26/07 - 01/08

On 29/07 after a delay we faced due to some financial issues we ordered our insert from IDT.

We also tried to design an insert with a scrambled miRNA, in order to use it as a control in our experiments, but because of its complexity we were not able to order it.

We started thinking of how our wiki should be written, which links should be included and what would the basic design of it look like.

On 01/08 we spotted a mistake on the design of our insert concerning the Signalling Peptide of Lamp-2b, so the insert which was ordered was wrong.

02/08 - 08/08

August 2021

Our plasmid backbone arrived on 03/08 from Addgene. So we programmed a bacterial culture training with the staff of the Laboratory as our insert had not yet arrived.

We carefully redesigned our insert and moved on to a new order on 04/08.

09/08 - 15/08

We went to the laboratory to observe bacterial transformation and streaking in LB-agar plates(09/08), single colony liquid inoculation(10/08), Plasmid DNA mini preps (11/08), Restriction enzymes digestion (to test the enzymes available on the laboratory) (12/08), Gel electrophoresis (13/08), performed by the PhD candidate and our advisor Christos Eftathiou. On 14-15/08 our wet lab team studied the protocols in order to move on and amplify the plasmid which we received from Addgene.

16/08 - 22/08

Our Wet Lab team went to the laboratory on 16/08 and poured 10 LB-agar plates with (100μg/ml) Ampicillin, in order to streak the bacteria containing the plasmid from addgene. On 17/08 our team stroke bacteria on 2 LB-agar+Amp plates from the bacterial stab received from addgene. On 18/08 LB-broth was made and later on the same day single colonies were selected and inoculated in 10ml of LB-broth. On 19/08 mini preps were performed in order to purify the plasmid, also our insert arrived from IDT. On 20/08 the purified plasmid was run in an 1% agarose gel to verify that it was normally purified. We needed to test its concentration on a nanodrop in order to proceed with restriction enzyme digestions, but the nanodrop in our department facilities was not available until the 25/08.

23/08 - 29/08

Due to some restrictions our team could not visit the laboratory. Thankfully we were able to perform the nanodrop quantification on 25/08.

At this period we were able to focus more on our Human practices and organize some events that would be held later such as the Bio-girls webinars, the blood donation and the Lab on the street.

30/08 - 05/09

We were constantly trying to organize our Human Practices oriented events by contacting several local clubs. We also prepared for our participation in ΔΕΘ which would be held on 11-17/09. We designed a banner, leaflets and presentations of our team for that purpose.

Our wet Lab team tried to test the performance of the restriction enzymes available in our laboratory under several conditions. On 30/08 NheI, XbaI, KpnI, BamHI and HindIII were tested on 37oC overnight. The digested plasmid was visualized under UV after agarose gel electrophoresis on 01/09. On 05/09 the same enzymes were put for digestion overnight using different buffers.

06/09 - 12/09

September 2021

The digested DNA from 05/09 was visualized under UV after agarose gel electrophoresis on 06/09. On 09/09 the same enzymes were put in digestion overnight and they were visualized on 10/09 under UV after agarose gel electrophoresis. The only enzymes digesting adequately at 37oC overnight were HindIII from Takara (M buffer). So, based on our design, we needed XbaI and NheI as well. The order was made on 10/09.

13/09 - 19/09

On 15/09 XbaI arrived but NheI would delay even more. So we had to borrow NheI from a nearby lab for that purpose. But the NheI that we borrowed was from NEB (buffer 2.1) while HindIII was from Takara (M buffer), so since we wanted to do a double digestion with HindIII and NheI they needed to perform on the same buffer. For that reason, we had to test NheI on M buffer. Therefore, we put NheI for digestion on 18/09 at 37oC overnight and visualized the results under UV after agarose gel electrophoresis on 19/09.

20/09 - 26/09

The results of the digestion were ambiguous so we decided to test NheI again on 21/09 at 37oC overnight and visualized the results on 22/09. Because a high amount of DNA was digested and later loaded, the bands on the agarose gel were also not very well interpreted. Therefore on 22/09 we put another digestion and on 23/09 we verified that it was working properly. On the same day, we measured our insert concentration on the Nanodrop. On 24/09 we put our plasmid for a double digestion with HindIII and XbaI and our insert for a double digestion with HindIII and NheI. On 26/09 we put the digested DNA (plasmid and insert) for ligation and we run the ligated mix on an agarose gel so that we could extract the wanted ligated part.

27/09 - 03/10

On 27/09 we performed the gel extraction protocol so that we could purify the plasmid ligated with our insert and measured it on the nanodrop, but the results were not very promising. We also met with our PI on that day who encouraged us to run the experiment from the beginning and perform the gel extraction before the ligation, to isolate the vector of the plasmid without the part which was cut during digestion.

On 28/09, with the remaining DNA from the digestion on 24/09, we performed the gel extraction protocol to isolate our vector without the part cut during digestion. On 29/09 we used the SpeedVac to increase the concentrations of our vector and insert and put them for ligation overnight. We also put our plasmid and insert for another double digestion on the same day. We also poured 4 plates with LB-agar + Amp (100μg/ml).

On 30/09 we performed transformation on DH5alpha E.coli with the ligation mix of 29/09 and streaked the transformed bacteria on 2 LB-agar plates. We extracted the digested plasmid (29/09 digestion) and then left them for overnight ligation with our insert (29/09 digestion). 200ml of LB broth were also made on the same day.

On 01/10 we picked and inoculated single colonies (29/09 ligation mix) in liquid cultures and left overnight in 5ml of LB broth with ampicillin (100μg/ml). On the same day we performed the transformation with the ligation mix from 30/09 and streaked 2 more LB-agar plates + Amp (100μg/ml).

On 02/10 we performed plasmid purification mini preps on the liquid cultures from the ligation of 29/09 and measured them on the nanodrop. On the same day, single colonies were picked and inoculated on 5ml liquid LB broth + Amp (100μg/ml) and left at 37oC overnight.

On 03/10, the purified DNA from 02/10 went through an agarose gel electrophoresis but no DNA was found on the gel. We also purified the plasmid DNA from the liquid cultures of the 30/09 ligation mix and ran an agarose gel electrophoresis. Some colonies were found to have some DNA and we inoculated them on 25ml of LB-broth in order to perform midi preps the next day.

04/10 - 10/10

October 2021

On 04/10 we realized that the colonies were inoculated without Ampicillin and therefore we decided not to proceed with those colonies. Therefore, we picked new colonies from both the ligation mix of 29/09 and 30/09 to inoculate in LB-broth + Amp(100μg/ml) and incubate at 37oC overnight.

On 05/10 we performed mini preps from the colonies we picked on 04/10 and ran the purified plasmid DNA on an agarose Gel. Some colonies seemed to have the plasmid with our insert so we inoculated these colonies in 25ml LB-broth + Amp(100μg/ml) in order to perform midi preps on the next day.

On 06/10 we performed 5 mini preps instead of midi preps for each colony, due to lack of resources. The DNA was purified and measured on the nanodrop.

On 07/10 we met with our PI and she advised us to digest the purified DNA with a restriction enzyme in order to make sure it was the plasmid with the insert. So, we put the purified DNA on digestion with HindIII and left it overnight.

On 08/10 we run the digested DNA on an agarose gel but unfortunately no visible DNA was found.

On 09/10 we held the Lab on the street event in Alexandroupolis.

On 10/10 we put another digestion of the purified DNA with HindIII in case any procedure went wrong on 07/10.

11/10 - 17/10

On 11/10 we ran the digestion on an agarose Gel but again we did not see what we expected.

On 12/10 we tried one last time with KpnI instead of HindIII and ran the DNA on an agarose gel. No DNA was visible. Therefore, we picked new colonies from the LB-agar plates from 30/09 and 01/10 and inoculated them in 5ml LB broth.

On 13/10 unfortunately we had to inoculate the bacteria in fresh LB broth + Amp (100μg/ml) because we were not able to perform the minipreps on that day.

On 14/10 we performed the DNA minipreps on the colonies and 4 fresh LB agar + Amp (100μg/ml) plates in order to proceed with a new transformation the next day.

On 15/10 the purified DNA from 14/10 was measured on nanodrop. 2 colonies went through a digestion with KpnI and the digested DNA was ran in an agarose gel. On the same day, another transformation was performed with both the 29/09 and 30/09 ligation mix and we streaked 4 LB-agar + Amp(100μg/ml).

On 16/10 single colonies were picked from the previous transformation.