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:
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1st Cycle
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| 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.
There were two main types of reactions:
1) Heterodimer association:
π[ππΉ-π·ππ΄]/ππ‘ = ππ β [ππΉ] β [π·ππ΄] β ππ β
[ππΉ-π·ππ΄]
Which describes the binding and dissociation of a given
transcription factor (TF) to its corresponding DNA molecule,
therefore forming a complex.
2) State transition: π[π]/ππ‘ = ππ β
[ππΉ-π·ππ΄]
Which describes the formation of a protein, induced by its
corresponding transcription factor (TF) β gene(DNA) complex
For specific information of how the model functions you can go
to the DryLab/Genetic Circuit Model page of our wiki.
The remaining equations were mainly to describe protein
degradation
d[aa]/dt = [P]* k_deg
Where any given protein (P) would have a constant degradation
rate (k_deg) over a set time period
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| 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.
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| 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.
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NF-kB Genetic Circuit v01
NF-kB Genetic Circuit v01 Simulation results
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2nd Cycle
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| 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 βlostβ 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.
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| 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.
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| 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.
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| 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.
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NF-kB Genetic Circuit v02.
NF-kB Genetic Circuit v02 Simulation results 1 of 2
NF-kB Genetic Circuit v02 Simulation results 2 of 2
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1st Cycle
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| Design |
The second model we built was the βExosome Production
Modelβ. 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.
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| Build |
To make this model we used two types of equations
representing a State Transition and a Heterodimer
Association/Disassociation.
The two equation types are represented as such:
1) State Transition: d[RNA or P]/dt = kst* [RNA or P]
2) Heterodimer Association: d[Exosome]/dt = ke* [RNA] *
[P]
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| 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.
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| 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.
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Exosome Production Model v01
Exosome Production Model v01 Simulation results
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2nd Cycle
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| 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.
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| 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 β [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]
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| 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.
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| 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.
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Exosome Production Model v02
Exosome Production Model v02 Simulation results
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1st Cycle
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| 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.
Plasmid design: pcDNA3 GFP LIC cloning vector (6D) was
chosen and ordered from Addgene.
Insert design: as described in the Design page and ordered
as a gene fragment from IDT.
The digestions and ligation were performed in silico using
Geneious.
The experimental design for developing the
construct:
β’ Amplification of the plasmid backbone
β’ Suspension of the Gene Fragment from IDT
(insert)
β’ Insert double digestion (HindIII, NheI)
β’ Plasmid double digestion (HindIII, XbaI)
β’ Plasmid and Insert Ligation
β’ Bacteria Transformation
Experiments to assess if the construct is
assembled:
β’ Diagnostic Digestions
β’ Gel electrophoresis
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| 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/ΞΌl
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/ΞΌl. So, in total we had about 500ng of
our insert.
Digestions and ligation: 7.5ΞΌl of the insert digestion
(37.5ng of 2.7kb) and 4.5ΞΌl of the plasmid digestion (50ng
of 5kb) were ligated
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| Test |
We ran 10ΞΌl 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)
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| 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.
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Plasmid design
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2nd Cycle
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| 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.
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| 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.
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| 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).
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| 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
βtranslatesβ 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.
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