iGEM 2021 — Team Greece_United

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# Team:Greece United/Notebook

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* HOME
* PROJECT

  [DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Description)
  [IMPLEMENTATION](https://2021.igem.org/Team%3AGreece_United/Implementation)
  [ENGINEERING](https://2021.igem.org/Team%3AGreece_United/Engineering)
  [RESULTS](https://2021.igem.org/Team%3AGreece_United/Results)
  [IMPACT](https://2021.igem.org/Team%3AGreece_United/Impact)
  [CONTRIBUTION](https://2021.igem.org/Team%3AGreece_United/Contribution)
  [PARTNERSHIP](https://2021.igem.org/Team%3AGreece_United/Partnership)
  [GRAPHICS](https://2021.igem.org/Team%3AGreece_United/Graphics)
* WET LAB

  [DESIGN](https://2021.igem.org/Team%3AGreece_United/Design)
  [EXPERIMENTS](https://2021.igem.org/Team%3AGreece_United/Experiments)
  [NOTEBOOK](https://2021.igem.org/Team%3AGreece_United/Notebook)
  [PART COLLECTION](https://2021.igem.org/Team%3AGreece_United/Part_Collection)
* DRY LAB

  [MODEL](https://2021.igem.org/Team%3AGreece_United/Model)
  [APPLICATION](https://2021.igem.org/Team%3AGreece_United/Model/App)
  [GENETIC CIRCUIT](https://2021.igem.org/Team%3AGreece_United/Model/Circuit)
* HUMAN
  PRACTICES

  [HUMAN PRACTICES](https://2021.igem.org/Team%3AGreece_United/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3AGreece_United/Education)
  [COMMUNICATION](https://2021.igem.org/Team%3AGreece_United/Communication)
  [COLLABORATIONS](https://2021.igem.org/Team%3AGreece_United/Collaborations)
  [INCLUSIVITY](https://2021.igem.org/Team%3AGreece_United/Inclusivity)
* ENTREPRENEURSHIP

  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship)
  [MARKET
  ANALYSIS](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/MarketAnalysis)
  [STRATEGIC
  PLANNING](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/StrategicPlanning)
  [PRODUCT
  DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/ProductDescription)
* TEAM

  [TEAM](https://2021.igem.org/Team%3AGreece_United/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3AGreece_United/Attributions)[SPONSORS](https://2021.igem.org/Team%3AGreece_United/Sponsors)
* MEDALS

* [November 2020](#heading1)

* [December 2020](#heading2)

* [January 2021](#heading3)

* [February 2021](#heading4)

* [March 2021](#heading5)

* [April 2021](#heading6)

* [May 2021](#heading7)

* [June 2021](#heading8)

* [July 2021](#heading9)

* [August 2021](#heading10)

* [September 2021](#heading11)

* [October 2021](#heading12)

# 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](https://2021.igem.org/Team%3AGreece_United/Design).

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](https://2021.igem.org/Team%3AGreece_United/Engineering).

---

# 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.](https://2021.igem.org/Team%3AGreece_United/Education#heading7)

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.

---

---

---

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# Team:Greece United/Engineering

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  [DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Description)
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  [MODEL](https://2021.igem.org/Team%3AGreece_United/Model)
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* ENTREPRENEURSHIP

  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship)
  [MARKET
  ANALYSIS](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/MarketAnalysis)
  [STRATEGIC
  PLANNING](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/StrategicPlanning)
  [PRODUCT
  DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/ProductDescription)
* TEAM

  [TEAM](https://2021.igem.org/Team%3AGreece_United/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3AGreece_United/Attributions)[SPONSORS](https://2021.igem.org/Team%3AGreece_United/Sponsors)
* MEDALS

* [Overview](#heading0)

* [Genetic Circuit Model](#heading1)

* [Exosome Production Model](#heading2)

* [Genetic construct assembly](#heading3)

# Overview

Throughout the project we practiced good engineering and research principles strictly conforming
to standards and ethics.
Here we present the engineering cycle for three major components:

### 1. Genetic Circuit Model

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **1st Cycle** | |  |  | | --- | --- | | Design | In order to construct the genetic circuit, we first had to think about the different species (DNA, Protein molecules) we had to add and think about how they would interact with each other (architecture of the model). So, we wrote down a list of the species, found the appropriate equations that describe their interactions and accurate biological constants which should be added to the respective equations. | | Build | We then started building the circuit in the CellDesigner software. The circuit consisted mainly of Genes, Proteins and complexes that interact with each other. After that we added the reactions and equations that describe each biological interaction. | | Test | We ran simulations on the built in CellDesigner simulator and interpreted the results.   After a few test-runs we analyzed our results and found out that there are many things wrong with our circuit design. During long periods of time our circuit did not stop being expressed which is something that does not conform with reality. Furthermore, we got some negative values, like that of the sensor, which was problematic. | | Learn | These results could be interpreted as if there was something wrong in our architecture, equations or something was missing entirely.    We concluded that we had to change some of our values and equations, in order to make our circuit more accurate and applicable to real world scenarios. | | ![](https://static.igem.org/mediawiki/2021/0/03/T--Greece_United--Eng01.png) |
| **2nd Cycle** | |  |  | | --- | --- | | Design | This time around we decided to change a few of our equations and correct the mistakes we made in others. Also, we decided to add another species to the circuit, which we named “lost” and it represents the destruction of a gene, due to cell division. This ensured that our results would be more accurate during long periods of time. | | Build | We added a reaction to each of the genes and gene-protein complexes to signify cell dilution.   The equation we used was: d[lost]/dt = [Gene] \* kdil   In addition, we corrected some of our equations and fixed the values of some constants that were outright incorrect and this is the result. | | Test | After we finished building the circuit, we ran a few simulations for different time periods, which led us to the results on the right.    In the second version of the circuit, not only are there no negative values, but we managed to make it run accurately over long time periods. The only species the value of which does not become 0 is the NF-kB. The reason is that we did not add a degradation equation to it, because during inflammation there is a constant supply of NF-kB being produced, therefore adding it would mess up our results. | | Success | In conclusion, we developed a genetic circuit that can accurately calculate the expression its effector for different values of NF-kB. The results provided by this model, helped us determine the value of adding such a circuit to our project and gave us knowledge on how we can correctly model biological pathways. | | ![](https://static.igem.org/mediawiki/2021/7/70/T--Greece_United--Eng02a.png)  NF-kB Genetic Circuit v02.      ![](https://static.igem.org/mediawiki/2021/c/cd/T--Greece_United--Eng02.png)  NF-kB Genetic Circuit v02 Simulation results 1 of 2   ![](https://static.igem.org/mediawiki/2021/c/c7/T--Greece_United--Eng02b.png)  NF-kB Genetic Circuit v02 Simulation results 2 of 2 |

### 2. Exosome Production Model

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **1st Cycle** | |  |  | | --- | --- | | Design | The second model we built was the “Exosome Production Model”. This model starts with a single plasmid that produces an RNA molecule. This RNA molecule is sub-divided into two new RNA molecules, the pre-miRNA and the mRNA, which codes for the Lamp2b-CAP protein. The pre-miRNA and the Lamp2b-CAP are then loaded into exosomes, which are the product of the model. | | Build | To make this model we used two types of equations representing a State Transition and a Heterodimer Association/Disassociation. | | Test | We ran the model and interpreted the results.   Certain values, like that of the RNA Complex, remained unchanged throughout the simulation time, something that is not intended to happen. The results from this model were all over the place, mainly because we had a bad architecture design. | | Learn | A few things had to change for the next iteration of the Exosome Production Model. First, we had to change the way the exosomes are produced from a two step to a one step reaction. Secondly, we needed to add another type of exosome that did not contain the Lamp2b-CAP protein. We learned from our literature that even though these exosomes have a lower synthesis rate, that rate is significant, and they needed to be factored in. | | ![](https://static.igem.org/mediawiki/2021/7/71/T--Greece_United--Eng03.png)  Exosome Production Model v01       ![](https://static.igem.org/mediawiki/2021/7/74/T--Greece_United--Eng03a.png)   Exosome Production Model v01 Simulation results |
| **2nd Cycle** | |  |  | | --- | --- | | Design | The design of the 2nd iteration of the Exosome Production Model was more of an overhaul, since we kept only a small number of things from the previous one. We added a transcription factor that binds to the gene, the complex of which activates the synthesis of the RNA molecules. Also, we added the new type of exosomes, that only contains the pre-miRNA, and changed the production of both exosomes to take only a single step, instead of two. The last things we changed was the removal of a reaction that converted the RNA Complex to mRNA, skipping the production of the pre-miRNA. | | Build | We kept the equation types of the previous iteration of the model, and added one more, that being the Reversible Heterodimer Association, that helps form the transcription factor-gene complex.    The three equation types are represented as such:   1) Reversible Heterodimer Association: d[TF-GENE Complex]/dt = [TF]\*[GENE]\*ka – [TF-GENE Complex]\*kd    2) State Transition: d[RNA or P]/dt = kst\* [RNA or P]    3) Heterodimer Association: d[Exosome]/dt = ke\* [RNA] \* [P] | | Test | We ran the model and interpreted our results.   The results of the 2nd version of the Exosome Production Model were more accurate and representative of the real world. The two types of exosomes were synthesized as expected from our literature and during long operations, all values eventually approached to 0. | | Success | In conclusion, the model we created can calculate the number of exosomes synthesized, with and without the Lamp2b-CAP protein. The importance of this model was significant, because the results given could us aid to design a more effective solution to tackle the problem of osteoarthritis. | | ![](https://static.igem.org/mediawiki/2021/1/1d/T--Greece_United--Eng004.png)  Exosome Production Model v02      ![](https://static.igem.org/mediawiki/2021/9/98/T--Greece_United--Eng04a.png)  Exosome Production Model v02 Simulation results |

### 3. Genetic construct assembly (part:BBa\_K3840312)

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **1st Cycle** | |  |  | | --- | --- | | Design | The purpose of the part is to be transfected in HEK293T cells and be expressed constantantly. For that, we needed to assemble a genetic construct, to be able to perform the transfection. | | Build | Plasmid Amplification: The plasmid was received in a bacterial stab and was amplified. The purified plasmid DNA was measured on Nanodrop giving a concentration of 110ng/μl while the 260/280 & 260/230 ratios were 2.00 & 2.12, meaning that the isolated DNA was of high purity.   Suspension of the Gene Fragment: The Gene Fragment was received dry and we had to resuspend it. We measured the resuspended insert on Nanodrop and we had a concentration of approximately 5 ng/μl. So, in total we had about 500ng of our insert.   Digestions and ligation: 7.5μl of the insert digestion (37.5ng of 2.7kb) and 4.5μl of the plasmid digestion (50ng of 5kb) were ligated | | Test | We ran 10μl of the ligation mix in an agarose gel to see if the ligation has worked.    Electrophoresis showed that the plasmid was ligated in many different combinations, and we could not spot the construct that we designed (vector,5kb + insert, 2.7kb = 7.7kb) | | Learn | After the digestion of the plasmid, the gene of GFP was left with HindIII and XbaI sticky ends. Which means that it could ligate with both the plasmid and the insert, making many combinations possible in the ligation. So, we had to change our design concerning the digestions & ligations. | | ![](https://static.igem.org/mediawiki/2021/9/9b/T--Greece_United--Eng05a.png)  Plasmid design       ![](https://static.igem.org/mediawiki/2021/a/ac/T--Greece_United--Eng05d.png) |
| **2nd Cycle** | |  |  | | --- | --- | | Design | To surpass this problem we thought of performing a gel extraction after the digestion of the plasmid. This way, we would be able to spot the band in an agarose gel which corresponds to the GFP gene that we do not need and causes the problems in the ligation.    Also, considering that the insert is about half the size of the vector and that a normal molar ratio of insert to vector ratio is about 3:1, we thought of increasing the amount of insert in the ligation reaction. | | Build | We put our plasmid for digestion exactly as the previous time and we performed gel extraction.    We condensed our samples using Speedvac, so that we can use more DNA in the ligation reaction and finally achieved the desired ratio of 1:4 vector to insert.   The next step was to transform the bacteria with these 2 ligation mixes and eventually we performed plasmid purification preps to isolate the plasmid DNA. | | Test | We measured each sample on Nanodrop and then performed diagnostic digestions followed by a gel electrophoresis.    We tested about 50 bacterial colonies. Electrophoresis showed no DNA (the only visible bands are ladder bands and control bands of ordered plasmid). | | Learn | Based on our measurements, the most probable reason that the genetic construct was not observed, is because of problems in the plasmid purification preparations.    We believed that because the bacteria survived in the LB even though Ampicillin was added in all the transformations that we tried. Theoretically, even in the case of an infection, the other bacteria should not be able to survive. Therefore, we should have chosen more wisely the kit we used or try some other kits or plasmid purification protocols to isolate the plasmid DNA.    A way to answer this question would be to choose a plasmid backbone which enabled us for a blue/white screening at the stage of the colonies selection. In that way, we would be able to discern the colonies that were equipped with the insert. So, if we noticed after the blue/white screening colonies that had the insert, we would conclude that the problem was indeed during the plasmid DNA purification. In the case that there were no colonies with the insert we should rethink some things concerning the ligation process. This approach would also help us to make our experiments more time efficient, which means more experiments which “translates” to more results.    Another weak aspect of our design is that we decided to order our insert from IDT as a gene fragment. The problem with that is that a very low amount of DNA was available from our insert to use in the ligations. We should have tried to order our insert in a high copy vector, which would enable us to amplify it limitlessly. A 3rd engineering cycle is about to begin. | | ![](https://static.igem.org/mediawiki/2021/a/a5/T--Greece_United--Eng06a.png) |

---

---

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# Team:Greece United/Experiments

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* ENTREPRENEURSHIP

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  [MARKET
  ANALYSIS](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/MarketAnalysis)
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  PLANNING](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/StrategicPlanning)
  [PRODUCT
  DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/ProductDescription)
* TEAM

  [TEAM](https://2021.igem.org/Team%3AGreece_United/Team)
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* MEDALS

In order to implement our design, we had to perform certain experiments. Our project consisted of
2 basic experimental parts. The Genetic construct assembly and the transfection of HEK293T in order
to measure the effects that our construct caused. We only managed to perform the experiments for the
1st part of our experimental design. You can find all of the protocols we used in our project below:

| Protocols |
| --- |
| IDT gene Fragment suspension (1000ng) | **Materials**   -  IDT Gene Fragment   -  Water for Injection     **Equipment**   -  Incubator at 50 ℃   -  Mini centrifuge   -  Vortex mixer     **Protocol**   -  Spin down the IDT gene Fragment tube before opening it in the mini centrifuge.   -  Add 100μl of water for injection   -  Vortex briefly.   -  Incubate at 50 ℃ for 15-20 min.   -  Vortex briefly.   -  Spin down the minicentrifuge. |
| Double digestions | **Materials**   -  DNA to be digested   -  Enzymes to be used   -  A common buffer for the 2 enzymes   -  Water for Injection     **Equipment**   -  Microcentrifuge tubes   -  Incubator at 37 ℃     **Protocol**   -  Add the 1μg of DNA in a microcentrifuge tube.   -  Add 5μl of buffer.   -  Add 1μl of each enzyme (10units).   -  Add water for injection to a final volume of 50μl.   -  Incubate at 37 ℃. The incubation time highly depends on the enzyme. |
| Gel extraction | **Materials**   -  Agarose gel with DNA that is desired to be exctracted   -  QIAquick Extraction Kit   -  Buffer GQ   -  Isopropanol   -  3M sodium Acetate     **Equipment**   -  QIAquick spin column   -  Laboratory blade   -  Blue Light source   -  Scale   -  2ml Microcentrifuge tube   -  Incubator at 50 ℃     **Protocol**   -  Place the agarose gel over a blue light source   -  Excise the gel slice containing the desired DNA band with a clean laboratory blade.   -  Place the gel slice in a microcentrifuge tube.   -  Tare the scale with an empty microcentrifuge tube.   -  Weigh the gel slice. Maximum 400mg for 1 QIAquick column.   -  Add 3 volumes of buffer GQ to 1 volume of 1% agarose gel. (300μl for 100mg of gel). For 2% agarose gel add 6 volumes of buffer GQ.   -  Incubate the tube at 50 ℃ for approximately 10 min (until gel is fully dissolved. Vortex every 2-3 min to help the gel dissolve.   -  Check the color of the mixture, it should be yellow.   -  If it is not Yellow (orange or violet), add 10 μl of 3M Sodium Acetate and mix.   -  Add 1 volume of isopropanol and mix (100μl for 100mg of 1%gel)   -  Place a QIAquick spin column in a 2 ml collection tube.   -  Add the dissolved gel to the QIAquick spin column.   -  Centrifuge for 1 min at 17,900 x g (13,000 rpm) and discard the flow through.   -  Add 500μl of buffer GQ to the column and centrifuge for 1 min at 17,900 x g (13,000 rpm). Discard the flow-through.   -  Add 750μl of buffer PE into the column, let the buffer in the column for 5 min and then centrifuge for 1 min at 17,900 x g (13,000 rpm). Discard the flow-through.   -  Centrifuge for 1 min at 17,900 x g (13,000 rpm) to remove residual buffer PE.   -  Place the column in a microcentrifuge tube.   -  Add 30μl of Elution Buffer (buffer EB) to the center of the column. Let stand for 4 min. Centrifuge for 1 min at 17,900 x g (13,000 rpm).   -  The DNA from the agarose gel slice that was cut is purified. |
| SpeedVac Vacuum Concentration | **Materials**   -  Sample for condensation     **Equipment**   -  SpeedVac Vacuum Concentrator   -  Microcentrifuge tube     **Protocol**   -  Measure the volume of the sample.   -  Load the same volume of water in another microcentrifuge tube.   -  Place the tubes on the concentrator antidiametrically.   -  Select centrifugation time depending on the volume of the sample and the desired final volume and start the centrifugation.   -  Measure the volume of your sample and if it is not the desired repeat the process. |
| Ligation | **Materials**   -  Digested plasmid DNA   -  Digested insert DNA   -  T4 DNA ligase   -  T4 DNA ligase buffer     **Equipment**   -  Microcentrifuge tube     **Protocol**   -  Add a maximum of 18 μl DNA. The plasmid to insert ratio is usually 1:3 so the amount of DNA depends on the plasmid and insert size.   -  Add 2μl of T4 DNA Ligase buffer.   -  Add 1μl of T4 DNA ligase.   -  If needed, add water for injection for a final reaction volume of 20μl.   -  Let the reaction proceed overnight at RT (25 ℃). |
| 10 LB agar+Amp(100μg/ml) plates | **Materials**     -  Pre-mixed LB-agar powder   -  Ampicillin stock solution (50mg/ml)   -  ddH2O     **Equipment**     -  Autoclave   -  Durant flask (500ml)   -  Heat resistant gloves   -  Microwave oven   -  10 Petri dishes (60 mm x 15 mm)   -  Ice bucket   -  Scale   -  Sterilization tape   -  Working flame     **Protocol**     -  Fill the Duran flask with 120ml of dd H2O   -  Measure 4.4 g of premixed LB-agar powder (37g for 1L)   -  Dissolve the LB-agar powder in the Duran Flask   -  Heat the flask without the lid in the microwave oven to fully dissolve the LB-agar powder. Avoid boiling the solution. Use a heat resistant glove for this step   -  Place the lid on the flask. Leave it half open.   -  Place a sterilization tape on top of the lid   -  Autoclave the solution in the autoclave for 30 min at 37 ℃, 20psi with the lid half open. The sterilization tape should darken by the end.   -  Spray and wipe the laboratory bench in which the plate pouring will be held with 70% ethanol. Label 10 petri dishes and stack them on your bench.   -  Let the dissolved LB-agar temperature drop to 60℃. To speed up the process place the side of the flask under running water and mix gently. Do not let any of the water touch the neck or top of the flask as this water is not likely to be sterile. Avoid letting the solution for too long as the LB-agar might solidify.   -  Place the ampicillin stock(50mg/ml) in your ice bucket.   -  Light a working flame in the place the LB-agar will be poured.   -  Add 240μl of Ampicillin(50mg/ml) in the flask. Close the flask and mix gently to spread ampicillin evenly.   -  Pour the solution in one plate, enough to cover 80% of the surface of the dish. Close the dish and swirl the plate to cover all of the surface.   -  Repeat for the remaining plates.   -  Leave the plates on the bench to solidify and dry overnight on the bench. |
| LB broth | **Materials**     -  LB broth powder   -  ddH2O     **Equipment**     -  Autoclave   -  Durant flask (500ml)   -  Heat resistant gloves   -  Microwave oven   -  Scale   -  Sterilization tape     **Protocol**     -  Fill the Duran flask with 200ml of ddH2O   -  Measure 4 g of premixed LB-agar powder (20g for 1L)   -  Dissolve the LB-broth powder in the Duran Flask   -  Heat the flask without the lid in the microwave oven to fully dissolve the LB-agar powder. Avoid boiling the solution. Use a heat resistant glove for this step   -  Place the lid on the flask. Leave it half open.   -  Place a sterilization tape on top of the lid.   -  Autoclave the solution in the autoclave for 30 min at 37℃, 20psi with the lid half open. The sterilization tape should darken by the end.   -  Let the LB-broth cool down and fully close the lid.   -  Any antibiotic can be added at this stage. |
| DH5 alpha transformation with Ampicillin resistance plasmid | **Materials**   -  Competent DH5a bacteria   -  DNA   -  LB-agar+Amp(100μg/ml)   -  LB broth     **Equipment**   -  Shaking incubator at 37 °C   -  Stationary incubator at 37 °C   -  Water bath at 42 °C   -  Ice bucket filled with ice   -  Microcentrifuge tubes   -  Sterile spreading device   -  Pasteur Pipettes   -  Working Flame     **Protocol**   -  Spray and wipe the laboratory bench with 70% ethanol.   -  Thaw the competent cells (taken from -80) on ice for approximately 30 min.   -  Place LB-agar+Amp plates at 37℃ or at RT (25℃).   -  Light a working flame on the bench.   -  Mix 1-10 DNA (1-100ng) into 20-100 μl of competent cells in a microcentrifuge tube. Mix gently.   -  Let the mixture on ice for 30 min.   -  Place 1/3 to 2/3 of the microcentrifuge tube in the water bath (42℃) for 45 sec.   -  Place the tube back on ice for 2 min.   -  Add 1ml of LB broth and incubate in a shaking incubator for 30-45 min at 37℃.   -  Pour 50μl of transformed cells on a LB-agar+Amp(100μg/ml) plate.   -  Hold the tip of a Pasteur Pipette on the working flame for ~5 sec until it moltens to seal it. Molten the Pasteur Pipette 2-3cm from the tip until it bends ~90°.   -  Streak the bacteria on the plate with the Pasteur pipette.   -  Centrifuge the rest of the transformation mix for 1-2 min at ~8.000 rpm to pellet the rest of the bacteria.   -  Resuspend the bacteria by pipetting.   -  Pour the resuspended on another LB-agar+Amp(100μg/μl) plate and repeat the procedure.   -  Incubate the plates overnight at 37℃ with the plates upside down (the lid on the down side). |
| Bacteria Inoculation in LB-broth+Amp(100μg/ml) | **Materials**   -  LB broth   -  Ampicillin stock (50mg/ml)   -  LB-agar+Amp(100μg/ml) cultures     **Equipment**   -  5ml Falcon tubes   -  Sterile tips   -  Shaking incubator at 37℃   -  Pipette filler   -  Sterile serological pipettes   -  Ice bucket filled with ice     **Protocol**   -  Spray and wipe the laboratory bench with 70% ethanol.   -  Light a working flame on the bench.   -  Label the falcon tubes that are going to be used.   -  Fill the falcon tubes with 5 ml of LB broth.   -  Add 10μl of Ampicillin stock(50mg/ml) in each falcon tube.   -  Pick a single colony from the LB-agar+Amp(100μg/ml) culture using a sterile tip and let the tip inside a falcon tube containing LB broth+Amp(100μg/ml).   -  Close the falcons with the lid Half way through and stabilize the lid using tape.   -  Incubate the liquid cultures overnight at 37℃ at 200 rpm. |
| QIAprep Spin Miniprep Plasmid DNA Purification | **Materials**   -  QIAspin Miniprep kit   -  Buffer P1   -  Buffer P2   -  Buffer N3   -  Buffer PE   -  Elution Buffer (Buffer EB)   -  Liquid Culture of bacteria in LB-broth     **Equipment**   -  QIAprep 2.0 spin column   -  Centrifuge   -  Microcentrifuge tips     **Protocol**   -  Centrifuge the bacterial cultures at 8.9rpm (6800 g) for 3 min at room temperature.   -  Discard the supernatant.   -  Resuspend the pellet with 250μl of buffer P1. Vortex if needed. Transfer each bacteria culture in a microcentrifuge tube.   -  Add 250μl of buffer P2 and mix by inverting the tubes 4-6 times. Do not allow for the lysis reaction to proceed for more than 5 min.   -  Add 350μl of buffer N3 and mix immediately by inverting the tube 4-6 times.   -  Centrifuge for 10min at 13.000rpm(~17,900 g).   -  Transfer 800μl of the supernatant in a QIAprep 2.0 spin column.   -  Centrifuge for 1 min at 13.000rpm(~17,900 g) and discard the flow through.   -  Add 750μl of buffer PE. Centrifuge for 1 min at 13.000rpm(~17,900 g) and discard the flow through.   -  Centrifuge for 1 min at 13.000rpm(~17,900 g) to remove residual wash buffer.   -  Place the QIAprep 2.0 column in a clean microcentrifuge tube.   -  Add 50μl of Elution Buffer (Buffer EB) to the center of the QIAprep 2.0 column, let stand for 1 min and centrifuge for 1 min at 13.000rpm(~17,900 g).   -  The isolated purified DNA is collected in the microcentrifuge tube diluted in Elution Buffer to a final volume of 50μl. |
| Nanodrop 2000 DNA concentration measurement | **Materials**     -  ddH2O   -  DNA sample   -  Solution that DNA sample is diluted (blank sample)     **Equipment**     -  NanoDrop 2000 Spectrophotometer   -  Paper towel   -  Laboratory wipe     **Protocol**     -  Open the Nanodrop 2000 softeware on the connected PC. Select “DNA” and then enter the wanted units in which the concentration is desired to be measured.   -  Lift the arm of the spectrophotometer and pipette 2μl of ddH2O onto the bottom pedestal.   -  Lower the arm. Lift the arm and gently touch the pedestals with the laboratory wipe.   -  Lift the arm and pipette 1 μl of the blank sample on the bottom pedestal.   -  Lower the arm and click on “Blank” on the computer.   -  Lift the arm and gently touch both pedestals with the laboratory wipe.   -  Pipette 1 μl of DNA sample on the bottom pedestal and lower the arm.   -  Click on “Measure”.   -  Save the archive.   -  Lift the arm. Gently touch the laboratory wipe on both pedestals.   -  Pipette 2 μl of ddH2O on the bottom pedestal and lower the arm.   -  Lift the arm and gently touch the laboratory wipe on both pedestals. |
| 1% agarose gel electrophoresis (50ml) | **Materials**   -  Agarose basic powder   -  TBE 1x   -  Ethidium Bromide stock (10mg/ml)   -  6x Loading Dye   -  DNA ladder   -  DNA sample     **Equipment**   -  Casting Tray   -  Well combs   -  Microwave oven   -  Gel box   -  Voltage source   -  Heat resistant gloves   -  100ml flask   -  scale     **Protocol**   -  Prepare the casting trey and place the well combs.   -  Pour 50ml of TBE in a 100ml flask.   -  Measure 0.5g of agarose basic.   -  Dissolve 0.5g of agarose basic in the flask.   -  Using heat resistant gloves dissolve the agarose on the TBE by heating it in the microwave oven. Avoid boiling the solution.   -  Cool the solution by placing the flask under running water and. swirling gently. Do not cool the solution too much because the gel will solidify.   -  Add 2μl of Ethidium Bromide in the solution and mix gently.   -  Pour the solution in the casting trey and leave in RT(25℃) to solidify for approximately 30 min.   -  Remove carefully the well combs.   -  Place the gel in the gel box with its wells on the side of the negative pole of the gel box.   -  Fill the gel box with TBE until all of the gel is covered.   -  Dilute 5 μl of loading Dye with 25μl of DNA Sample in a microcentrifuge tube.   -  Carefully Load the DNA samples and Ladder in the wells of the gel.   -  Connect the gel box with the Voltage source.   -  Set the Voltage to 80-120 volts.   -  Observe the dye on the gel.   -  When the dye has nearly reached the edge of the gel, you can take the gel out of the gel box and observe it under UV light. |

---

---

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# Team:Greece United/Results

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* HOME
* PROJECT

  [DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Description)
  [IMPLEMENTATION](https://2021.igem.org/Team%3AGreece_United/Implementation)
  [ENGINEERING](https://2021.igem.org/Team%3AGreece_United/Engineering)
  [RESULTS](https://2021.igem.org/Team%3AGreece_United/Results)
  [IMPACT](https://2021.igem.org/Team%3AGreece_United/Impact)
  [CONTRIBUTION](https://2021.igem.org/Team%3AGreece_United/Contribution)
  [PARTNERSHIP](https://2021.igem.org/Team%3AGreece_United/Partnership)
  [GRAPHICS](https://2021.igem.org/Team%3AGreece_United/Graphics)
* WET LAB

  [DESIGN](https://2021.igem.org/Team%3AGreece_United/Design)
  [EXPERIMENTS](https://2021.igem.org/Team%3AGreece_United/Experiments)
  [NOTEBOOK](https://2021.igem.org/Team%3AGreece_United/Notebook)
  [PART COLLECTION](https://2021.igem.org/Team%3AGreece_United/Part_Collection)
* DRY LAB

  [MODEL](https://2021.igem.org/Team%3AGreece_United/Model)
  [APPLICATION](https://2021.igem.org/Team%3AGreece_United/Model/App)
  [GENETIC CIRCUIT](https://2021.igem.org/Team%3AGreece_United/Model/Circuit)
* HUMAN
  PRACTICES

  [HUMAN PRACTICES](https://2021.igem.org/Team%3AGreece_United/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3AGreece_United/Education)
  [COMMUNICATION](https://2021.igem.org/Team%3AGreece_United/Communication)
  [COLLABORATIONS](https://2021.igem.org/Team%3AGreece_United/Collaborations)
  [INCLUSIVITY](https://2021.igem.org/Team%3AGreece_United/Inclusivity)
* ENTREPRENEURSHIP

  [ENTREPRENEURSHIP](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship)
  [MARKET
  ANALYSIS](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/MarketAnalysis)
  [STRATEGIC
  PLANNING](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/StrategicPlanning)
  [PRODUCT
  DESCRIPTION](https://2021.igem.org/Team%3AGreece_United/Entrepreneurship/ProductDescription)
* TEAM

  [TEAM](https://2021.igem.org/Team%3AGreece_United/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3AGreece_United/Attributions)[SPONSORS](https://2021.igem.org/Team%3AGreece_United/Sponsors)
* MEDALS

* [Wet Lab](#heading1)

* [Dry Lab](#heading2)

* [Survey](#heading3)

# Results

## Wet Lab

During the our lab experiments we tried to assemble our genetic construct. Unfortunately,
we were not able to transfect HEK293T cells in order to end up with some results concerning the
part that we had designed, as it was not successfully inserted in a plasmid backbone.

Below, we can see 3 different agarose gels(1%) in which we performed gel electrophoresis. 15 colonies of
DH5a transformed with the ligation mix were tested to see if any of these colonies had the plasmid we desired.

So, after plasmid DNA purification for each colony picked, we ran the agarose gel electrophoresis to be able to observe
the DNA isolated. Unfortunately, there are no visible bands of the plasmid we desired in any of the colonies.

![](https://static.igem.org/mediawiki/2021/a/ab/T--Greece_United--wl-re-1.png)

**Figure 1:**Electrophoresis gel, colonies 1-6

On the first column we can see the ladder spreading evenly and on the 8th column our vector was used as a
control. In columns 1-6 the isolated DNA from the colonies was run. There are some pale band in columns 1 , 2 and 3.

![](https://static.igem.org/mediawiki/2021/b/b2/T--Greece_United--wl-re-2.png)

**Figure 2:**Electrophoresis gel, colonies 1-12

On the first column we can see the ladder spreading evenly and on the 8th column our vector was used as a control.
In columns 7-12 the isolated DNA from the colonies was run. There are some pale visible bands in columns 8, 11 and 12.

![](https://static.igem.org/mediawiki/2021/b/b9/T--Greece_United--wl-re-3.png)

**Figure 3:**Electrophoresis gel, colonies 13-15

On the first column we can see the ladder spreading evenly and on the 8th column our vector was used as a control.
In columns 13-15 the isolated DNA from the colonies was run. We can see some very pale bands in columns 14 and 15.

Colonies 1,8 and 11 were further grown and their plasmid DNA was Isolated again. This time diagnostic digestions
were performed and the results follow:

![](https://static.igem.org/mediawiki/2021/2/27/T--Greece_United--wl-re-4.png)

**Figure 4:**Agarose gel - Digested with HindIII

![](https://static.igem.org/mediawiki/2021/f/ff/T--Greece_United--wl-re-5.png)

**Figure 5:**Agarose gel - Digested with KpnI

Figure 4: In this gel the isolated DNA and control were digested with HindIII.
We can see the ladder spread evenly on the 1st column and the control on the 5th column.
On columns 1, 8 and 11 the DNA isolated from the colonies 1,8 and 11 were ran. There is no visible plasmid DNA.

Figure 5:In this gel the isolated DNA and control were digested with KpnI. We can see the ladder spread
evenly on the 1st column and the control on the 5th column. On columns 1, 8 and 11 the DNA isolated from the
colonies 1,8 and 11 were ran. There is no visible plasmid DNA.

Therefore, we can surely conclude that our genetic construct was not successfully built.

# Results

## Dry Lab

### Approach A

We ran our simulation for a time span of 24h.

![](https://static.igem.org/mediawiki/2021/9/91/T--Greece_United--Figure2-1em.png)

**Figure 7:** CAP and regular exosomes
synthesized
with [TF] = 24000

Even though our model produces both CAP exosomes and regular exosomes, we can see that in 24h
the
amount of CAP exosomes synthesized
is far greater than that of regular exosomes (Figure 5). This is a desired result, because it
has
been proven that CAP exosomes have
higher affinity for chondrocytes, they get absorbed quickly by the cartilage cells and rarely
end up
entering the blood stream.
But why is there such a gap between these two types of exosomes?

To find out, we thought it would be best to see the difference in the expression rates of pre
miRNA
and the Lamp2b-CAP membrane protein.

![](https://static.igem.org/mediawiki/2021/4/47/T--Greece_United--Figure2-2em.png)

**Figure 8:** Lamp2b-CAP and premiRNA synthesis
with
[TF] = 24000

In Figure 6 we can see that there is indeed a higher amount of Lamp2b-CAP produced in the cells.
This result makes sense, because when the gene is transcripted once, we get at most a single
premiRNA molecule and an mRNA
molecule which is likely translated more than once. Over the spam of 24h this adds up to having
a
higher level of the membrane
protein and thus more CAP exosomes.

### Approach B

Below you can see all the graphs that resulted from our simulation.

![](https://static.igem.org/mediawiki/2021/c/c7/T--Greece_United--model-g1.png)

**Figure 9:** Graph of mRNA, miRNA and
protein rate through time.

![](https://static.igem.org/mediawiki/2021/f/f7/T--Greece_United--model-g3.png)

**Figure 10:** Graph of miRNA in exosomes and
Protein in exosomes value through time.

![](https://static.igem.org/mediawiki/2021/2/27/T--Greece_United--model-g4.png)

**Figure 11:** Graph of miRNA useful value
through time.

For a total runtime of **2880 minutes (= 2 days)** the cell produced, **16153920**
useful exosomes (that carry the protein and miRNA),
the total concentration of miRNA in the cell is: **[miRNA] = 298,527 CPC**, a total concentration of miRNA-
useful (miRNA that got into exosomes): **[miRNA useful] = 520,230,000 CPC**
and a total concentration of protein **[protein] = 755,747 CPC**, were produced by one cell
transfected with one plasmid.

The mean average of cells in a joint is 149,000,000 with a deviation of 46,000,000 cells. We used that
along with our results to calculate how much
concentration of miRNA would be in each cell, considering that the exosomes would reach
every cell equally. The average is **[miRNA] = 3.49148**
copies per cell while using the deviation the best-case scenario is **[miRNA] = 5.05078** copies per
cell and the worst-case scenario is **[miRNA]= 2.66785**
copies per cell.

**Concentrations are counted in copies per cell (CPC).**
Literature data have showed that exosomes with the guiding tag do not leave the target
joint, while without, can be found in the whole organism.
So, we calculated the effect exosomes with the protein will have inside the joint.

For a hypothetical introduction of N cells transfected with the plasmid into a joint, we
calculated the large-scale extent of idea, to simulate
how it would work and what efficiency it would have in more real-life conditions and
numbers. The calculations are similar to those above.
The results are presented in the table below.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| **N (cells)** | **Total [miRNA] (CPC)** | **Total [protein] (CPC)** | **Total [miRNA useful] (CPC)** | **Average case (CPC)** | **Worst case (CPC)** | **Best case (CPC)** |
| 1000 | 2.98955e+08 | 7.55747e+08 | 5.2023e+11 | 3491.48 | 2667.85 | 5050.78 |
| 2000 | 5.97911e+08 | 1.51149e+09 | 1.04046e+12 | 6982.96 | 5335.7 | 10101.6 |
| 3000 | 8.96866e+08 | 2.26724e+09 | 1.56069e+12 | 10474.4 | 8003.54 | 15152.3 |
| 4000 | 1.19582e+09 | 3.02299e+09 | 2.08092e+12 | 13965.9 | 10671.4 | 20203.1 |
| 5000 | 1.49478e+09 | 3.77874e+09 | 2.60115e+12 | 17457.4 | 13339.2 | 25253.9 |

Those results are promising and show that by introducing our therapy in its current design,
cells will be affected in the whole target
joint by a sufficient number of exosomes that will introduce a significant amount of miRNA
in each cell since miRNA copies per cell typically
vary between hundreds and 120000 copies per cell.

From literature we know that chondrocytes in osteoarthritis have lower levels of miRNA140
and many positive effects of increasing it have already
been documented by promoting cartilage formation and inhibiting its degeneration.
Even though we were not able to further correct our model due to lack of laboratory results,
it shows us that based on literature we have
**sufficient therapeutic potential.**

# Probing Public Opinion:

## The NOUS Survey

To probe the diversity of perspectives about the science of biology and synthetic biology
held by the public and consider these in the design of respective solutions (Science Communication
Goal #5)
we designed a field survey.

 Main objectives were:- a)  to assess the public's understanding of key biological concepts, and
- b)  to gather views on general policy issues related to synthetic biology

The field survey was approved by the Research Ethics Committee of Democritus University of Thrace
(approval no. 12/22-07-2021).

The survey comprised of a questionnaire with 2 parts. Each question was designed to be either FALSE
or TRUE and the participants
could answer using a 5-point Likert scale: Strongly Disagree, Disagree, Don’t know, Agree, Strongly
Agree.

The section about general understanding of biological
concepts
consisted of the following questions.

- **1.**   DNA contains information about the structure and
  function of the
  organism to which it belongs.
- **2.**   RNA is synthesized based on DNA.
- **3.**   Plants are living organisms.
- **4.**   RNA can modify DNA.
- **5.**   All organisms on Earth have a common ancestor.
- **6.**   Some antibiotics are effective against viruses.
- **7.**   Human somatic and gamete (eggs, sperm) cells have the
  same DNA.
- **8.**   Mushrooms are plants.
- **9.**   Genetically modified organisms (GMOs) are used to make
  drugs.
- **10.**   DNA fragments of genetically modified organisms we
  consume can modify
  or become integrated into the DNA of human cells.
- **11.**   The use of stem cells for research or therapeutic
  purposes requires
  the killing of embryos.

The second section of the survey contained questions on
the general
public’s opinion on policy issues of synthetic biology. The questions were:

- **1.**   A genetically modified organism or cell is any
  organism or cell whose
  genetic material has been modified using genetic engineering techniques. I
  understand this
  definition.
- **2.**   Genetic modification of organisms or cells is, by
  definition, immoral.
- **3.**   Consumption of any genetically modified organism (GMO)
  is proven to be
  dangerous to health.
- **4.**   The use of GMOs for drug production is a beneficial
  practice.
- **5.**   Genetic modification of human somatic cells for the
  treatment of
  diseases could be beneficial (cell changes are not inherited in the
  offspring).
- **6.**   Genetic modification of human somatic cells for the
  treatment of
  diseases is dangerous and should not be investigated.

The survey was advertised via the team’ssocial media and the questionnaires were handed out to the
public
in major team events, including the [85th HELEXPO
2021
International Fair in Thessaloniki](https://2021.igem.org/Team%3AGreece_United/Communication/TIF), Greece and in
Blood Donation Clinics.

During the questionnaire administration, participants were informed that the correct questions to
the answers
(part 1 of the survey) will be officially released via the team’s social media and planned
educational events.

Demographics of the participants, results and analysis of the 277 responses received are shown
below.
Both genders were represented (56% women). The majority were adults, 63% in the age group 18-40 and
23% in
the age group 40-65 years. The majority (75%) were of a higher education level and most likely in
the basic or medical
sciences (60%). Higher education students were 51% while 44% were employed at the time.

Main findings per questions are shown in the slideshow below:

![](https://static.igem.org/mediawiki/2021/e/e5/T--Greece_United--ppt--survey--overall.png)

![](https://static.igem.org/mediawiki/2021/7/78/T--Greece_United--survey--1.png)

![](https://static.igem.org/mediawiki/2021/a/a2/T--Greece_United--survey--2.png)

![](https://static.igem.org/mediawiki/2021/1/1d/T--Greece_United--survey--3.png)

![](https://static.igem.org/mediawiki/2021/0/0c/T--Greece_United--survey--4.png)

![](https://static.igem.org/mediawiki/2021/6/63/T--Greece_United--survey--5.png)

![](https://static.igem.org/mediawiki/2021/9/91/T--Greece_United--survey--6.png)

![](https://static.igem.org/mediawiki/2021/8/8a/T--Greece_United--survey--7.png)

![](https://static.igem.org/mediawiki/2021/7/78/T--Greece_United--survey--8.png)

![](https://static.igem.org/mediawiki/2021/f/f9/T--Greece_United--survey--9.png)

![](https://static.igem.org/mediawiki/2021/2/28/T--Greece_United--survey--10.png)

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