Team:Greece United/Engineering


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.

There were two main types of reactions:

1) Heterodimer association: 𝑑[𝑇𝐹-𝐷𝑁𝐴]/𝑑𝑑 = π‘˜π‘Ž βˆ™ [𝑇𝐹] βˆ™ [𝐷𝑁𝐴] βˆ’ π‘˜π‘‘ βˆ™ [𝑇𝐹-𝐷𝑁𝐴]
Which describes the binding and dissociation of a given transcription factor (TF) to its corresponding DNA molecule, therefore forming a complex.

2) State transition: 𝑑[𝑃]/𝑑𝑑 = π‘˜π‘  βˆ™ [𝑇𝐹-𝐷𝑁𝐴]
Which describes the formation of a protein, induced by its corresponding transcription factor (TF) – 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

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.

NF-kB Genetic Circuit v01

NF-kB Genetic Circuit v01 Simulation results

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 β€œlost” 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.

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

2. Exosome Production Model

1st Cycle
Design The second model we built was the β€œExosome 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.

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]

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.

Exosome Production Model v01

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.

Exosome Production Model v02

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.

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
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

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)
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.

Plasmid design

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 β€œtranslates” 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.