Intro

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Our project is centered around the production of the secondary metabolite decursin. The system includes the three penultimate reactions in the original synthesis pathway of decursin. This involves two enzymatic reactions and one chemical reaction. This means, in all, our project would involve four unique metabolites, umbelliferone, 7-demethylsuberosin, decursinol, and finally decursin. In order to assess if our reactions proceeded successfully, we needed a reliable and efficient method of detecting and quantifying our molecules while being capable of differentiating between various other metabolites in our system.

Usually, the state-of-the-art method used for such a need is high-performance liquid chromatography (HPLC)^[1] due to its sensitivity to various metabolites and ability to quantify said molecules. However, we quickly realized that while HPLC was a satisfactory solution from a technical standpoint, there were many downsides that made HPLC less than optimal. Firstly, we had many different designs on the synthetic biology side that would lead to a vast amount of HPLC runs in order to compare the various designs and arrive at a conclusion. Secondly, HPLC is a low throughput device, each run is time consuming, expensive and the running of the machine requires specific expertise^[2]. And thirdly, the HPLC device itself is very expensive and not widely accessible. These disadvantages deter iGEM groups from pursuing worthwhile projects as mentioned on our Human Practices page.

Due to all these reasons, we have developed a measurement tool for quantifying metabolites. We named it OraCell since it can assist in giving a clearer picture of the future of our project as well as other similar projects. Our method employs Luciferase as a reporter gene, and in the presence of the molecule of interest it registers a change in bioluminescence signal. As a side note, in Hebrew the word “Or” means light and since we’re utilizing changes in luminescence this made the choice of name even more appropriate.

The Hippo Pathway

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While researching decursin, we discovered that in addition to being a leading candidate in treatment for chemotherapy-induced alopecia, it significantly assists in the inhibition of tumor growth^[3]. One possible mechanism for its tumor suppression capabilities is through the Hippo signaling pathway^[4]. In this pathway, Yap/Taz proteins translocate to the nucleus of mammalian cells, bind to a TEAD transcription factor which in turn binds to a specific DNA motif, inducing transcription of TEAD-regulated genes^[5]. In the presence of decursin, the upstream elements in the Hippo pathway are phosphorylated, inducing the destruction of Yap/Taz proteins, inhibiting their nuclear localization and reducing transcription^[6]. Following the revelation that decursin impacts an existing signaling pathway, we planned to incorporate the Hippo pathway in our method for quantifying and detecting decursin.

OraCell

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The OraCell system is based on an existing plasmid with the Luciferase reporter gene, downstream of TEAD response elements (Addgene plasmid #34615). Transfection of this plasmid yields a constitutive expression of the reporter gene. When adding decursin or any metabolite that interacts with the Hippo pathway to these cells, the TEAD transcription factor should be unable to bind to the DNA resulting in a decrease of the bioluminescence signal. Following consultations with professors knowledgeable in this subject in general and this plasmid specifically, we chose to use Chinese Hamester Overian (CHO) cells as the host organism.

We wanted to create a system in which the results will be easily reproducible. Since mammalian cells do not maintain plasmids, a transient transfection assay would require a secondary reporter under no regulation, by which the Luciferase signal would be normalized. Alternatively, the system could be integrated into the genome of our organism of choice, giving rise to a new cell line that is dedicated to researching the Hippo-pathway and the molecules that affect it. We decided to go with the latter, and integrated the plasmid into the genome of CHO cells by cloning a selection marker that confers resistance to blasticidin, a toxin of mammalian cells, under a CMV promoter. This will reduce the noise in our system and remove any unrepeatability between experiments.

To establish a cell line, CHO cells were transfected with our modified system. 24 hours post-transfection we changed the media to a version containing 8 ug/ml blasticidin. Cells that do not integrate the plasmid into their genome would dilute the plasmid as they divide, while cells that integrated the system would pass the locus down to the daughter cells. After a selection period of 7 days, cells were seeded in a single cell per well ratio using Fluorescence-activated Cell Sorting (FACS). Once propagated, we incubated clonal cell populations in each cell with luciferin, and Luciferase-positive cells were then established as the OraCell system. These cells were then capable of determining the concentration of decursin in any given sample. A duration of one hour incubation with decursin was found to be sufficient, the cells are then lysed and release of the existing Luciferase enzyme whose amount varies in a decursin-dependent manner. Luciferin is then added, which is catalyzed by the Luciferase enzyme, yielding bioluminescence that can be measured with a luminometer. This value can then be compared with a calibration curve determining the concentration of purified decursin in the original sample.

Figure 2: OraCell example animation

Experiments & Methods

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The main pillars of the workflow used to establish the “OraCell” stable cell-line are composed of multiple experiments and protocols devised based on knowledge provided by our mentors and our literature review.

Plasmid Construction

We used the 8xGTIIC-Luciferase plasmid as a backbone for our plasmid system (Addgene plasmid #34615). It includes a pGL3b vector and the genes encoding for Luciferase downstream of GTIIC motifs with high affinity for TEAD transcription factors. This was convenient due to the abundance of prior results. For our stable integration, we required an assembly of a constitutive CMV promoter with a blastR resistance gene (iGEM part BBa_K1943015)

Insert 1: CMV promoter

The constitutive mammalian CMV promoter, the CMV enhancer, and two restriction sites (MluI, HindIII) were amplified from a plasmid provided to us by our PI.

Insert 2: blastR

The blasticidin resistance gene blastR was amplified from a plasmid provided to us by our PI's lab. A KpnI site was added using primers we designed, alongside the existing HindIII site.

Assembly

The assembly was achieved with restriction of the backbone with mentioned restriction sites. Following the restriction, a 3-part ligation (backbone, CMV insert, blastR insert) was performed successfully to achieve the final construct. Results were validated using sequencing and colony PCR alongside gel electrophoresis.

Preliminary calibration experiments

Once we verified that our plasmid was ready to be transfected to the mammalian cells, we were able to continue to the next stage in the workflow.

We chose to work with CHO cells, because they are easily growable in large-scale cultures and demonstrate high stability and safety profiles.

Working with mammalian cells in the time span of this project required delicate planning. In accordance, preliminary experiments were conducted to get acquainted with the protocols of splitting, seeding and transfection, and find optimal parameters such as the time it takes for plates to reach confluence and the optimum concentration of the transfection reagents .

Seeding calibration

CHO cells were grown in 10cm plates, and experiments were performed in 6-well plates. Due to time constraints, an optimal seeding density of a 6-well plate was sought, such that overnight incubation would result in a confluent plate on the next day.

6-well plates were seeded with a different cell density ranging from 10⁴ cells /cm² to 10⁵ cells/cm². After overnight incubation we counted the number of cells in each plate using a counting chamber.

Linear Polyethylenimine (PEI, Mw 25kDa) Calibration

Our chosen method of transfection was PEI. This transfection reagent is used to introduce the plasmid DNA to the host cell, it condenses DNA into positively charged particles that are endocytosed and transferred to the cytoplasm. ^[8] PEI was chosen due to its availability and low cost. Stock solution concentration was 1mg/ml.

We conducted an experiment to find the optimum mass of PEI in transfection experiments. While PEI provides a way to introduce DNA into the cell, it is also toxic. An optimal amount would be high enough to transfect the highest percentage of cells possible, while retaining a reasonable percentage of viable cells.

Our experiment included a plasmid backbone with a Blue Fluorescent Protein (BFP) insert reporter gene. The BFP would be used to quantify the efficiency of the transfection; cells that underwent transfection would express BFP that in turn expresses fluorescence.

2.5 μg of BFP plasmid was mixed with increasing amounts of PEI and used FACS to measure the cell’s viability and fluorescence 48 hours post-transfection.

Stable Integration

To establish a stable clonal cell line, cells were transfected with the blasticidin resistance gene (BlastR)-modified Luciferase plasmid and subjected to a high concentration of blasticidin. Then, we used FACS (Bigfoot Spectral Cell Sorter) to perform single-sorting cell into individual wells on a plate and allowed cells to proliferate before testing their Luciferase expression with luciferin.

Luciferase Assay

To measure the transfected cell reporter gene, Luciferase, we performed a luminescence assay. This was done both to validate the transfection itself, and to study how decursin affects the gene expression.

We used a Biotium’s Firefly Luciferase Assay Kit 2.0, cells were lysed, and luciferin was added to the lysate. This resulted in an oxidation reaction that emitted a measurable signal. The signal was measured using the bioluminescent mode of a plate reader. Our positive control for the experiment was E. coli β-10 cells transfected with a p.lux plasmid that contains the genes encoding for both Luciferase and luciferin.

Results

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Our initial cloning, calibrations and luminescence experiments can be found on our results page. Following these preliminary results, we began adding decursin and measuring the luminescence to investigate if there was indeed a decrease. The first of these experiments was a time calibration experiment to see under which incubation time we get an optimal decrease in luminescence. The two negative controls used in the experiment, OraCell cells without luciferin and CHO cells that don’t express Luciferase, both displayed minimal luminescence in comparison to the positive control and experiment group (Figure 5). Additionally, the positive control, OraCell cells without decursin, following 22 hours incubation showed higher luminescence compared to OraCell cells that were incubated with decursin. This was a good indication that the system functioned as expected, in the presence of decursin, there was a decrease in the transcription of our reporter gene.

Figure 5 . Decursin incubation time calibration. OraCell cells were incubated with decursin with various time points ranging from 1-22 hours, lysed, luciferin was added and bioluminescence was measured. In addition, there were two types of negative control used, OraCell cells without luciferin added and CHO cells that do not express Luciferase. The positive control was OraCell cells incubated without decursin

This implied that with further testing of decursin concentrations, it would be possible to build a calibration curve of decursin concentrations as a function of luminescence. To our surprise, we noticed an unexpected trend in the experiment group, a periodic oscillation. The level of luminescence was low following a one-hour incubation and gradually increased until the peak around 12 hours, at which point the luminescence begins decreasing until it reaches a level roughly equivalent to the starting luminescence. We speculated that this could be due to the fact that the Hippo pathway is involved in cell proliferation and naturally oscillates during the cell cycle^[9]. This led us to pursue a two-fold experiment which consisted of the testing of various decursin concentrations at two different time points as well as the highest and zero concentrations at two additional time points.

Similar to the previous experiment (Figure 5), the OraCell cells incubated with decursin once again showed a periodic behavior (Figure 6). Following one hour of incubation time with decursin, the greatest decrease in luminescence in comparison to the cells without decursin was observed. The differences between the control group, without decursin, and the cells that were incubated with decursin gradually decreases until their luminescence was roughly equivalent following a ten-hour incubation, meaning that decursin had a minimal impact. This result fits nicely with the previous finding, that following a 12-hour incubation with decursin, we receive a minimal decrease in luminescence (Figure 5). An interesting phenomenon occurs following 16 hours of incubation with and without decursin. In the control group for the first ten hours, the luminescent signal stayed approximately the same but after 16 hours, there’s a marked decrease (Figure 5). Even more interesting is the fact that the OraCell cells following a 16-hour incubation with decursin, show a higher luminescence signal compared to the control. This indicates that decursin caused up-regulation of the expression of the Luciferase enzyme, contrary to our predicted mechanism. This point is illustrated further by looking at the calibration curve of the same experiment (figure 7).

Figure 6: Decursin incubation time. OraCell cells were incubated with and without decursin at various time points ranging from 1-16 hours, lysed, luciferin was added, and bioluminescence was measured. The cells without decursin served as the positive control, to see how much decursin impacted the cells’ bioluminescence.

The calibration curve experiment was conducted on two of the time points from the incubation time experiment, 3 and 16 hours. The OraCell cells were incubated with six different decursin concentrations, 0, 15, 30, 60, 120 and 240 µM for both time points. For the three-hour incubation, it is difficult to define a pattern. It appears that decursin causes mild fluctuations in the luminescence, both increasing and decreasing, compared to the cells without decursin, the zero concentration (Figure 7). The 16-hour incubation, on the other hand, showed a very clear trend, of higher luminescence with increasing decursin concentrations. Saturation was observed at the 60 µM concentration, at which point the decursin no longer observed to have an effect. Just as with the incubation time experiment, we see the opposite effect of what was expected. We expected that with increased decursin concentrations, luminescence will decrease; however, luminescence increased.

Figure 7: Dose response for decursin concentrations at 3 and 16 hours. OraCell cells were incubated with six concentrations of decursin ranging from 0-240 µM for two time points, 3 and 16 hours. The cells were then lysed, luciferin was added, and bioluminescence was measured. The luminescence was normalized to the zero concentration of decursin for each time point, respectively. Note that for the 16 h dose response, the trend shows a linear dependency.

Our working hypothesis to explain this phenomenon is that originally, we only partially understood decursin’s impact on the Hippo pathway. The Hippo pathway functions in two modes, Hippo on and Hippo off and these are both controlled by most of the same proteins^[10]. Thus, if decursin is capable of interfering with one mode of this pathway, it’s reasonable to assume that it can then interfere with the second mode as well. This means that if the OraCell cells are currently in the Hippo off mode, there are many upstream elements that interact with the Yap/Taz proteins, causing them to be dephosphorylated and cause expression^[11]. However, if the OraCell cells are currently in the Hippo on mode, there would also be other upstream elements that interact with the Yap/Taz proteins, causing them to be phosphorylated and inhibit expression^[11].

This would also explain why there’s a decrease in Luciferase expression in cells that didn’t receive decursin following the 16-hour incubation (Figure 6). The cells have made the switch to Hippo on, causing a natural decrease in expression, and therefore, when decursin is added, this process is interrupted giving an increase in Luciferase expression. This would also explain the unclear trend found following the three-hour incubation (Figure 7). It’s possible that the Hippo pathway is found in a limbo stage, between on and off, leading to conflicting signals and thus, decursin has a fluctuating effect. This phenomenon can be further investigated by adding more time points and adding changing decursin concentrations at each point as well as monitoring the Luciferase expression at each time point without the addition of decursin. Altogether, this would give a clearer picture of the natural fluctuations in Luciferase expression as a result of the Hippo pathways without decursin and if decursin is truly able to interfere with the Hippo pathway, regardless of the mode.

Prospects

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The OraCell assay was shown to be a efficient, simple and high-throughput method that holds the potential of detecting and quantifying a variety of molecules. The method still needs a few more experiments to verify its capabilities, which should hopefully be accomplished in the coming weeks to show its potential utility for groups all across the world.

This measurement could be repeated by other iGEM teams by preforming a simple Luciferase assay on the OraCell cell line. The protocols used were described in our Protocols page.

In future implementations, the OraCell cell line could be used not only as for quantification assays by other teams, but also to mass screen molecules for their effect on the Hippo Pathway. Compared to traditional YAP/TAZ assays that require re-transfection on each assay, OraCell provides a high-throughput and low-cost solution. Generating datasets of molecules related to the Hippo Pathway could have uses alongside machine learning algorithms for new drug discovery. The research of the Hippo Pathway, an important tumor suppressor^[12], could lead to advances in the field of cancer research and open the door for new treatment strategies.