Team:Greece United/Notebook
CSS_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.