Team:Greece United/Results
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.
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.
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.
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:
Figure 4:Agarose gel - Digested with HindIII
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.
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.
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.
Figure 9: Graph of mRNA, miRNA and protein rate through time.
Figure 10: Graph of miRNA in exosomes and Protein in exosomes value through time.
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:
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.
The second section of the survey contained questions on the general public’s opinion on policy issues of synthetic biology. The questions were:
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, 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:
