Engineering Cycle
Synthetic biology can be applied in various fields, such as healthcare, agriculture, industrial biotechnology, and many more. It is consequently important to have a structured approach to ensure the best solutions. The engineering cycle is applied specifically to optimize biological systems and organisms.
The engineering cycle has four stages: Design, Build, Test, and Learn. This can be seen in Figure 1.
Design: The first step is design. It begins with a brainstorming phase, where problems are identified. Then a lot of research is conducted to understand the problem and conceptualize possible solutions. The solution can then be visualized by sketching or modeling. In synthetic biology, the design could be a therapeutic compound or a biosensor.
Build: When the design is finalized, the next phase is to build it. This is the practical implementation of the design, where the product is constructed. An example could be the assembling of designed DNA sequences followed by cloning.
Test: When the product has been built, it needs to be tested. This is to ensure that the product functions as expected and meets all the requirements to solve the initial problem. The product can be tested to assess different parameters, such as functionality, durability, and stability.
Learn: After the testing, the results are analyzed to identify what worked and what was unsuccessful. The data is used to pinpoint exactly what can be improved in the design, construct or experimental conditions. This will enhance the product, ensuring an optimal solution to solve the problem (1).
When one cycle is completed, the newfound knowledge can be used to improve the design, thus starting the engineering cycle again.
Cycle A - Preliminary plasmid design
Cycle A consists of the preliminary designs for a non-hormonal, minimally invasive treatment for Endometriosis.
Design
We started by defining the problem: Endometriosis. A painful disease with a long list of symptoms, making everyday life a challenge for those affected. An even bigger issue is the lack of research and treatment methods. We came up with the idea to kill the Endometriosis cells, without affecting the alike, but healthy, endometrium cells. We started by researching the differences in normal endometrial cells and in endometriosis cells, to find a way to only target endometriosis cells.
We found out that in endometriosis cells the estrogen receptor beta (ERβ) is upregulated, and estrogen receptor alpha (ERα) is downregulated compared to endometrium cells (2). Studies have shown that in the endometriotic-like cells the estrogen receptor β (ERβ) becomes upregulated by >100 times (3).
The next thing we then had to Figure out was how we could use that knowledge to create something that could kill the endometriosis cells but leave the Endometrium cells unharmed. We knew that to kill a cell we could use an apoptosis-inducing protein, and that ER’s are nuclear receptors, meaning they can function as transcription factors. We began our search to find an apoptosis protein, that could be inhibited by another protein, as well as promotors which were regulated by ERα and ERβ.
We decided to design two different plasmids: Plasmid design 1, seen in Figure 2, and Plasmid design 2, seen in Figure 6. In plasmid design 1, we would use the concentration difference of both ERα and ERβ by using two different promoters, one activated by ERα and the other by ERβ. In plasmid design 2 we would only use the concentration difference of ERβ, where we would use a promoter activated by ERβ and a promoter inhibited by ERβ.
We designed two different plasmids because we had not yet decided which cell lines we were going to use. We thought about testing the plasmids on lung epithelial cells, as they were already available in the lab. The plan was to use estrogen in the media, in hope that the extra estrogen would change how much of each receptor that would be expressed, but this seemed unlikely to work. We also investigated the possibilities with using HeLa cells, as they have some similarities with endometriosis cells. Another possibility was engineering the lung epithelial cell line with lentivirus, so we could make some express more ERβ and some express more ERα, mimicking endometriosis and endometrium cells respectively. Our last possibility was to order endometrial cells and endometriosis cells.
After some research we narrowed the promoters down to the following:
CD47 promoter, which is activated by ERβ (4).
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BRCA1 promoter, which is activated by ERα (5).
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pS2/TFF1 promoter, which is activated by ERα and Activating Protein 1 Complex (AP1) (6).
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EGFR promoter, which is inhibited by ERβ (7).
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ERα promoter, which is inhibited by ERβ and activated by ERα (8).
After some discussion we decided to use the CD47 promotor for both plasmids, the ERα promoter for plasmid design 2 and the BRCA1 promoter for plasmid design 1.
To ensure that the promoters worked as intended we designed two control plasmids, where the apoptosis inducing protein and the apoptosis inhibitor are replaced with a red fluorescent protein (DsRed1) and green fluorescent protein (GFP). Control Plasmid 1 contains the same promoters as plasmid design 1, and Control Plasmid 2 contains the same promoters as plasmid design 2.
When searching for genes, we prioritized finding a gene for a protein inducing apoptosis, which also can be inhibited by another protein. We found the following:
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Tumor protein P53 (p53), inhibited by Mouse double minute 2 homolog (MDM2) (9).
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BCL2 Associated X (BAX), inhibited by B-cell leukemia/lymphoma 2 protein (BCL2) (10).
We did not choose the p53-MDM2 interaction, as it is involved in cancer development, and we wanted to decrease the possibility of the plasmid having negative side effects. Therefore, we decided to use BAX as our apoptosis protein and BCL2 as our inhibitor. In plasmid design 1 the CD47 promoter activates BAX, and the BRCA1 promoter activates BCL2. In plasmid design 2 the CD47 promoter activates BAX, and the ERα promoter activates BCL2. The next step was to find a terminator. We accomplished this by seeking assistance from our supervisors who recommended Simian virus 40 PolyA (SV40 Poly(A)), which we decided to use for our designs.
Now that we had figured out all our parts, we needed to find a suitable vector and develop a cloning strategy for the assembly. Upon request were able to get the pcDNA3.1(+) plasmid donated from Kim Ravnskjær, Associate Professor at SDU, and we used that as our backbone.
We used SnapGene to design our plasmids and together with our supervisors we decided to use Gibson Assembly to assemble our plasmids.
Design 1
Plasmid design 1 (pB1-BCL2-CD47-BAX), is the design with the BRCA1 promoter coupled to the BCL2 gene followed by the SV40 terminator, and the CD47 promoter coupled to the BAX gene followed by the SV40 terminator in the pcDNA3.1(+) vector, which can be seen in Figure 2.
When this plasmid is introduced in an endometriosis cell, the estrogen will bind to dimerized ERβ. This complex binds to the CD47 promoter, initiating the transcription and translation of the BAX gene. As the BAX protein is an apoptosis inducing protein, this will ultimately kill the cell. The effect of plasmid design 1 on the endometriosis cells can be seen in Figure 3.
The concentration of ERβ is low in healthy endometrium cells, but not zero. For that reason, there is a safety switch in the plasmid, where the high concentration of ERα will activate the BCRA1 promoter, leading to the expression of the BCL2 protein. BCL2 inhibits BAX, which will ensure the survival of the cell. The effect of plasmid design 1 on healthy endometrium cells can be seen in Figure 4.
Control Plasmid 1 (pB1-G-CD47-R) can be seen in Figure 5. The plasmid consists of the BRCA1 promoter coupled to GFP followed by the SV40 terminator, and CD47 promoter coupled to DsRed1 followed by the SV40 terminator in the pcDNA3.1(+) vector. The Control Plasmid is expected to have the same activation of CD47 promoter as in plasmid design 1 in endometriosis cells, where there will be produced red fluorescent protein. In healthy endometrium cells, the BRCA1 promoter will be activated, and there will be produced green fluorescent protein.
Design 2
Plasmid design 2 (pERα-BCL2-CD47-BAX), consists of the ERα promoter coupled to the BCL2 gene followed by the SV40 terminator, and the CD47 promoter coupled to the BAX gene followed by the SV40 terminator in the pcDNA3.1(+) backbone, which can be seen in Figure 6. This design will have the same effect in endometriosis cells as plasmid design 1: Dimerized ERβ complex will activate the CD47 promoter, inducing the expression of BAX protein, leading to apoptosis in the cells. The ERβ complex will also inhibit the ERα promoter, ensuring that no BCL2 is produced. The effect of plasmid design 2 on endometriosis cells can be seen in Figure 7. When the plasmid is introduced into healthy endometrium cells the ERα will form a complex as well, which activate the ERα promoter, leading to the production of the BCL2 protein, and the survival of the cells, as seen in Figure 8.
Control Plasmid 2 (pERα-G-CD47-R) can be seen in Figure 9. This plasmid consists of the ERα promoter coupled to GFP followed by the SV40 terminator, and the CD47 promoter coupled to DsRed1 followed by the SV40 terminator in the pcDNA3.1(+) vector. The expected outcome is activation of CD47 promoter in endometriosis cells, leading to the expression of red fluorescent protein. In healthy Endometrium cells, the ERα promoter will be activated, and there will be expressed green fluorescent protein.
We preferred plasmid design 1 (pB1-BCL2-CD47-BAX), as the expected outcome is a higher percentage of surviving healthy endometrium cells. Therefore, we decided to order an endometrial cell line (Human Endometrial Stromal Cells (HESC)) and an endometriosis cell line (12Z Human Endometrial Epithelial Cells), as this would be the best proof-of-concept. Control Plasmid 1 (pB1-G-CD47-R) will be called Control Plasmid 1 and Treatment Plasmid 1 (pB1-BCL2-CD47-BAX) will be called Treatment Plasmid going forward.
Table 1: Parts in Control Plasmid 1 and Treatment Plasmid 1.
| Name Composite Part | Composite Part | Name Basic Part | Basic Part | Type | Designers | Length |
|---|---|---|---|---|---|---|
| Treatment apoptosis part | BBa_K5253007 | CD47 promoter | BBa_K5253000 | Promoter | Anna Kristensen | 1000 bp |
| BAX | BBa_K5253002 | Coding | Anna Kristensen | 576 bp | ||
| SV40 terminator | BBa_K5253006 | Terminator | Anna Kristensen | 122 bp | ||
| Treatment safety-switch | BBa_K5253008 | BRCA1 promoter | BBa_K5253001 | Promoter | Anna Kristensen | 1000 bp |
| BCL2 | BBa_K5253003 | Coding | Anna Kristensen | 717 bp | ||
| SV40 terminator | BBa_K5253006 | Terminator | Anna Kristensen | 122 bp | ||
| Control red fluorescent | BBa_K5253009 | CD47 promoter | BBa_K5253000 | Promoter | Anna Kristensen | 1000 bp |
| RFP | BBa_K5253004 | Coding | Anna Kristensen | 681 bp | ||
| SV40 terminator | BBa_K5253006 | Terminator | Anna Kristensen | 122 bp | ||
| Control green fluorescent | BBa_K5253010 | BRCA1 promoter | BBa_K5253001 | Promoter | Anna Kristensen | 1000 bp |
| GFP | BBa_K5253005 | Coding | Anna Kristensen | 717 bp | ||
| SV40 terminator | BBa_K5253006 | Terminator | Anna Kristensen | 122 bp |
Build
We used Gibson assembly to construct the plasmids in the lab. The insertion sequence was cloned into the pcDNA3.1(+) expression vector using the NEB Hifi DNA Assembly Master mix (NEB # M5520). The plasmids were then transformed into NEB 5-alpha Competent E. coli cells (NEB #C2987) to produce multiple copies. To ensure the transformation was successful, we used colony PCR. As the vector has ampicillin resistance, this was used to select the transformed colonies. Then PCR was performed along with gel electrophoresis to ensure the insertion sequence had the right length. Lastly the plasmids were sequenced through Sanger Sequencing to ensure there were no mutations in the final products, before the testing.
Test and Learn
To test if our plasmids design worked, we preformed transfection with our plasmid and different cell lines, 12Z and HESC. We decided to begin with the Control Plasmid, as it would allow us to use flow cytometry to measure the fluorescent levels, making it possible to asses the transfection efficiency and the functionality of the promoters. If our plasmid and transfection were successful, we would expect the 12Z cells to express a red fluorescence, because of the higher concentration of ERβ in these cells. In the HESC cells we expected the cells to express a green fluorescence, because of the higher concentration of ERα. As shown in Figure 10, our expected results were not achieved. A detailed description can be found on the results page. After some discussions we concluded that either our plasmids had to many endotoxins, our cells did not respond well to the transfection, or the transfection process failed, as the plasmid transfection could not be confirmed through PCR of the transfected cells.
Redesign
Human Practices had an interview with Martin Rudnicki, Professor and Doctor in Gynecology and Obstetrics at OUH, Director of Minimal Invasive Surgery. He gave some insights to another approach to target the endometriosis cells. He informed, that progesterone receptors could be another potential target for our plasmids, as their intracellular concentrations in endometriosis cells are more extensively researched. This led us to research progesterone receptors’ molecular capabilities and how to implement this into our current Treatment Plasmid.
We learned that progesterone receptors are nuclear receptors that can act as transcription factors when activated. The levels of progesterone receptors (PR’s) are low in endometriosis cells compared to healthy Endometrial cells. There are two isoforms: PRA and PRB. There are very low levels of PRA in endometriosis cells, whereas there aren’t detected any PRB in endometriosis cells (11).
Therefore, it was possible to make another survival switch in the Treatment Plasmid, by inserting a promoter targeted by the PRB, as this will ensure, that a higher percentage of healthy endometrium cells will survive, while the plasmid still promotes apoptosis in endometriosis cells.
We learned that the PRB can activate the PTEN promoter (12). When it was time to choose genes, we again wanted to use an apoptosis inhibiting gene. This time, instead of using BCL2, we chose the X-linked inhibitor of apoptosis (XIAP) gene. This gene inhibits apoptosis downstream of BAX by inhibiting caspase 3, 7 and 9 (13). The design of this Treatment Plasmid, pB1-BCL2-CD47-BAX-PTEN-XIAP, can be seen in Figure 11.
To summarize the effect of the plasmid in endometriosis cells: ERβ will activate the CD47 promoter, activating the transcription of BAX, which ultimately leads to apoptosis of the sick cells. This can be visualized in Figure 12.
In the endometrium cells, the plasmid will have the following effect: The low levels of ERβ will ensure that only a small amount of BAX is produced. ERα will activate the BRCA1 promoter, activating the transcription of BCL2. BCL2 inhibits BAX and therefore apoptosis. PRB will activate the PTEN promoter and XIAP is transcribed. XIAP stops apoptosis downstream of BAX by inhibiting caspase 9 as well as caspase 3 and 7, which can be seen in Figure 13. This establishes an even stronger safety switch in the Treatment Plasmid, ensuring a higher survival rate in the healthy endometrium cells.
Cycle B – HESC optimization
Cycle B explores the optimization of growth conditions for Human Endometrial Stromal Cells (HESC) using the engineering cycle approach.
The optimization process was initiated due to consistent issues with our HESC cell lines taking over a week to reach sufficient confluency for experiments. This prolonged growth period significantly delayed the experimental timeline, prompting us to investigate potential improvements in the cell culture conditions.
By systematically testing various media formulations, we aimed to identify the optimal balance of nutrients, growth factors, and serum components that would enhance cell proliferation and reduce the time needed for the cells to reach confluency. Through this process, we hoped to streamline the culture conditions, allowing for more efficient cell preparation and ultimately improving the overall workflow of our experiments.
Design
In this phase, we aimed to optimize HESC growth by testing various culture media. The focus was to explore how different components, particularly the types of serum and the use ofcharcoal-stripped FBS, influenced the confluency of HESC cultures. We tested six different media compositions to determine which conditions provided the best growth environment:
Table 2: This table gives an overview of the different media combinations used in the experiment. Each flask was assigned a code (e.g. “O2), corresponding to the type of media that they contain.
| Type | Conditions |
|---|---|
| Normal | DMEM-F12 + 10% charcoal-stripped FBS + 1% Pen-Strep + 2 mM L-Glutamine |
| O1 | DMEM-F12 + 10% Human AB Serum + 1% Pen-Strep + 2 mM L-Glutamine |
| O2 | DMEM-F12 + 10% charcoal-stripped FBS + 0.5% Pen-Strep + 2 mM L-Glutamine |
| O3 | DMEM-F12 + 10% FBS + 1% Pen-Strep + 2 mM L-Glutamine |
| O4 | Embryomax + 10% charcoal-stripped FBS + 1% Pen-Strep + 2 mM L-Glutamine |
| O5 | Embryomax + 10% Human AB Serum + 1% Pen-Strep + 2 mM L-Glutamine |
Build
The practical setup involved culturing HESC cells in each of the six media conditions in the design section. Cells were seeded at a consistent density in T25 culture flasks, ensuring that media composition was the only variable. Cultures were incubated under identical conditions, including temperature, humidity, and CO2 levels, and regular observations were made to assess cell confluency.
Test
The growth of the HESC cells was evaluated by monitoring the confluency of the cells in each condition every 1-2 days. This data was used to determine the growth rates - i.e. the percentage change in quantity the cells over a specified period of time - of the cells with the different media. The following results were observed:
Table 3: An overview of how much of a decrease in doubling time each media combination resulted in throughout the experiment.
| Type | Normal (T75) | Normal (T25) | O1 | O2 | O3 | O4 | O5 |
|---|---|---|---|---|---|---|---|
| Doubling time | 8.87 days | 6.24 days | 2.13 days | 2.55 days | 2.55 days | 1.78 days | 2.24 days |
The following graphs illustrate the growth patterns of cells cultured in Normal HESC cells (in a T25 flask) media and HESC cells grown in O3 media.
The significant difference in confluency progression highlights the enhanced growth rate observed in the O4 condition compared to the Normal T25 media. This aligns with the calculated growth rates, where O3 reached confluency much faster than the standard media.
These results provided insight into the optimal conditions for HESC growth based on media composition.
Learn
From our observations, the medium that yielded the highest growth rate was O3, O4, and O5, with growth rates of 0,8 days, 1,4 days, and 0,9 days (Table 3). In comparison the original composition used has a growth rate of 8,0 days for the T75 flasks and 3,0 days for the T25 flask.
Each of these formulations contains specific elements that may contribute to their greater performance. This medium provided the best balance of growth and cell health, likely due to factors such as the presence of fetal bovine serum (FBS), and the use of specialized EmbryoMax medium. These factors may explain the enhanced growth observed in these conditions compared to other tested media.
To better understand these results, we can break down the role of key components in the media and their potential effects on HESC proliferation:
FBS vs. Charcoal-stripped FBS vs. AB Serum:
- FBS (Fetal Bovine Serum): FBS is a rich source of growth factors, hormones, and nutrients (14), which makes it an ideal supplement for promoting cell growth. The fact that O3 contains FBS may explain why it performed well. FBS provides critical factors that promote cell proliferation, differentiation, and survival.
- Charcoal-stripped FBS: The charcoal stripping process removes hormones such as estrogen (15), which could affect the growth of HESCs. Since O4 contains charcoal-stripped FBS and still performed well, it suggests that the removal of hormones may have minimized certain unwanted interactions (e.g., excess estrogen) that could otherwise interfere with HESC growth. O4’s use of EmbryoMax medium may compensate for the lack of hormones by providing other essential growth factors.
- Human AB Serum: This serum is human-derived and may lack the breadth of growth factors found in FBS (16). However, O5 performed well despite using AB serum, possibly due to its use of EmbryoMax medium (which will be discussed further below). AB serum might more closely mimic the natural environment of human cells, reducing the variability seen with bovine-derived components.
DMEM/F-12 vs. EmbryoMax Medium
- DMEM/F-12 (Dulbecco's Modified Eagle Medium) is a common basal medium that supports a wide range of cells (17) but may not be fully optimized for HESC growth compared to a more specialized medium like EmbryoMax. This could explain why the Normal medium, which only uses DMEM/F-12, underperformed compared to O4 and O5.
- Both O4 and O5 used EmbryoMax medium, which is specially formulated to support the growth of embryonic stem cells (18). For HESCs, EmbryoMax may provide the right balance of nutrients and growth factors that enhance their growth potential.
- O4: The combination of EmbryoMax and charcoal-stripped FBS could explain why this medium performed well. While the charcoal-stripped FBS reduces hormone-induced stress, the EmbryoMax medium compensates with optimal growth conditions.
- O5: The presence of EmbryoMax medium with AB serum may have created a more physiologically relevant environment for HESCs, allowing better growth than media containing DMEM alone.
These factors combined suggest that the specialized components of O3, O4, and O5 closer mimic the optimal conditions for HESC growth compared to the normal, or other media tested. Moving forward, we plan to optimize the medium further by testing additional variables, to see if the growth rate can be further enhanced.
