iGEM 2023 — Team iitchicago

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Past Semester Reports

Class of Fall 2021 [Report](https://static.igem.wiki/teams/4896/wiki/f21-report.pdf).

Class of Spring 2022 [Report](https://static.igem.wiki/teams/4896/wiki/s22-report.pdf).

Class of Fall 2022 [Report](https://static.igem.wiki/teams/4896/wiki/f22-report.pdf).

Class of Spring 2023 [Report](https://static.igem.wiki/teams/4896/wiki/s23-report.pdf).

# August

### Week 1:

* Introduce project to new semester of students

### Week 2:

* Introduce iGEM to new semester of students

# September

### Week 3:

###### Vector Design Team

* Investigate genetic inverter gates

### Week 4:

###### Vector Design Team

* Develop goals for the Fall 2023 semester

###### Wet Lab Team

* Develop goals for the Fall 2023 semester
* Start lab training

###### Regulatory Response Team

* Develop goals for the Fall 2023 semester
* Start lab training

### Week 5:

###### Vector Design Team

* Investigate techracyline’s role in gene expression
* Identify molecules unique to the cholesterol biosynthesis pathway
* Study genetic switches in the liver

###### Wet Lab Team

* Finish lab training
* Isolate GFP DNA from E.coli

![](https://static.igem.wiki/teams/4896/wiki/gfp-dna.jpg)

###### Regulatory Response Team

* Continue lab training
* Inventory lab supplies

### Week 6:

###### Vector Design Team

* Research the tet-on and tet-off system
* Investigate genetic inverter gates in mammalian cells

###### Wet Lab Team

* Transfection optimization of GFP DNA in HepG2 cells

![](https://static.igem.wiki/teams/4896/wiki/transf-op.png)

###### Regulatory Response Team

* Continue lab training
* Determine if the siRNA sequences with the highest knockdown scores on the VectorBuilder website for HMGCR, DHCR24, and MVD can be validated with data provided on ThermoFisher Scientific’s website

# October

### Week 7:

###### Vector Design Team

* Investigate peptides that could replace tetracycline in gene expression
* Develop a SRE promoter using the tet-off system

![](https://static.igem.wiki/teams/4896/wiki/pro-design.png)

###### Wet Lab Team

* Transfect HepG2 cells acclimated at standard acclimated at conditions with V2 DNA
Develop methods to transfect HepG2 cells acclimated at low and high cholesterol conditions with V2 DNA
Start selection for cells containing V2 in antibiotic G418

###### Regulatory Response Team

* Continue lab training

### Week 8:

###### Vector Design Team

* Continue develop a SRE promoter using the tet-off system
* Investigate repressor degradation

###### Wet Lab Team

* Continue selection for cells containing V2 in antibiotic G418

###### Regulatory Response Team

* Finish lab training
* Extract RNA from wildtype HepG2 cells

# Daily Maintenance

In order to ensure the vitality of a cell line, it is advisable to perform daily upkeep on HepG2 cells. This routine care comprises:

* Checking for any signs of contamination
* Capturing daily images of the cells
* Monitoring the cell confluency

In addition to these daily tasks, it is essential to provide the cells with periodic attention, which includes:

* Changing the cell culture media
* Performing cell splitting

By consistently attending to both daily and routine maintenance procedures, one can uphold the health of the cell line and accurately monitor cell growth. This diligence is crucial for experiments such as transfection, which is indispensable for the project.

![](https://static.igem.wiki/teams/4896/wiki/low.png)

HepG2 Cells in Low Cholesterol Condition

![](https://static.igem.wiki/teams/4896/wiki/standard.png)

HepG2 Cells in Standard Cholesterol Condition

![](https://static.igem.wiki/teams/4896/wiki/high.jpg)

HepG2 Cells in High Cholesterol Condition

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Version 1 (V1)

![V1 plasmid map](https://static.igem.wiki/teams/4896/wiki/v1-vector.png)

* **pSRE:** pCMV containing SRE elements
* **Nluc:** Nano luciferase under pSRE control for insert verification
* **CMV promoter:** constitutive promoter
* **Neo:** Neomycin resistance
* **Ampicillin:** Ampicillin resistance

The V1 vector contains our engineered SRE promoter and one luciferase gene.

The SRE promoter will act as a cholesterol biosensor. Cholesterol biosynthesis is regulated by the sterol regulatory element binding proteins (SREBP) system which contains sterol regulatory elements (SRE). These SRE elements were transferred into a cytomegalovirus (CMV) promoter creating an engineered pSRE promoter that acts as a cholesterol biosensor.

Luciferase is a reporter gene that will indicate if the promoter is on or off. One luciferase gene was added to V1 to validate the function of the SRE promoter.

The results from the assay were inconclusive. An improved validation method was added to V2.

## Version 2 (V2)

![V2 plasmid map](https://static.igem.wiki/teams/4896/wiki/v2-vector.png)

* **pSRE:** pCMV containing SRE elements
* **Luciferase:** Firefly luciferase under pSRE control for insert verification
* **CMV promoter:** constitutive promoter
* **Rluc:** Renilla luciferase under pSCMV control for insert verification
* **Neo:** Neomycin resistance
* **Ampicillin:** Ampicillin resistance

The V2 vector contains our engineered SRE promoter and two luciferase genes.

The SRE promoter will act as a cholesterol biosensor. Cholesterol biosynthesis is regulated by the sterol regulatory element binding proteins (SREBP) system which contains sterol regulatory elements (SRE). These SRE elements were transferred into a cytomegalovirus (CMV) promoter creating an engineered pSRE promoter that acts as a cholesterol biosensor.

Luciferase is a reporter gene that will indicate if the promoter is on or off. Two luciferase genes were added to V2 to perform the dual luciferase assay to validate the function of the SRE promoter.

The results of the dual luciferase assay showed that the pSRE is sensitive to changes in cholesterol levels. The pSRE is active at low cholesterol levels and inactive at high levels.

## Version 2.5 (V2.5)

The IITChicago team is working to develop vector V2.5. This version will contain a redesigned SRE promoter that will be activated at high cholesterol levels.

![V2.5 plasmid map](https://static.igem.wiki/teams/4896/wiki/v2-5-vector.jpg)

This is a validation vector that is being used by the regulatory response team to test different siRNA’s and their ability to inhibit cholesterol synthesis. The siRNA’s are targeting rate limiting steps in the cholesterol synthesis pathway. The team is quantifying how well each siRNA inhibits cholesterol synthesis to identify ideal candidate(s) to be included in the final vector.

![V2.5 circuit design on](https://static.igem.wiki/teams/4896/wiki/v2-5-circuit-design-promoter-on.jpg)
![V2.5 circuit design off](https://static.igem.wiki/teams/4896/wiki/v2-5-circuit-design-promoter-off.jpg)

Our initial circuit design was composed of a cholesterol sensor pSRE, and a response element, a siRNA that inhibited some gene in cholesterol biosynthesis. There are many genes that we are investigating; our team has so far validated an effective siRNA against SQLE, squalene epoxidase. In this fashion, the cell can sense cholesterol level and adjust cholesterol biosynthesis.
However, when we began to validate the sensor, we realized that our circuit worked in the wrong direction, inhibiting cholesterol synthesis when cholesterol is low and allowing it to continue when cholesterol is high.

## Version 3 (V3)

The IITChicago team is working to develop vector V3. This version will contain the pSRE cholesterol biosensor and siRNA cholesterol regulator.

![V3 circuit design low chol.](https://static.igem.wiki/teams/4896/wiki/v3-low-chol.jpg)
![V3 circuit design low to high chol.](https://static.igem.wiki/teams/4896/wiki/v3-low-to-high-chol.jpg)
![V3 circuit design high chol.](https://static.igem.wiki/teams/4896/wiki/v3-high-chol.jpg)

In our most current design, we added an inverter element between the sensor element (pSRE) and the response element (the siRNA) to invert the regulation of siRNA.

* In a low cholesterol condition, pSRE will be activated causing repressor X to be transcribed, which will bind to pX (the promoter for the siRNA sequence) and prevent the siRNA from being transcribed, allowing cholesterol synthesis to proceed.
* In a high cholesterol condition, pSRE will not be active, stopping expression of X which then allows pX to turn on, producing the siRNA and inhibiting cholesterol production.

The X protein is engineered to contain a degron, which are tunable elements providing various degradation rates. This allows us to tune the degradation rate to eliminate X rapidly enough when pSRE is off, but allow it to accumulate when pSRE is on. We are currently investigating various X repression systems (e.g. TET, PIT) and degron elements.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## Cell Maintenance

Cell maintenance is crucial for ensuring the health, viability, and consistent morphology of cells in experiments. For HEPG2 cells, which are human liver carcinoma cells, proper maintenance ensures that they retain their characteristic metabolic functions. Regular monitoring and maintenance of the cells ensures that they are free from contamination, which can compromise the experiment, and instead ensures reproducibility of experimental data, allowing for reliable comparisons.

## Cell Splitting

Cell splitting, or subculturing, is a vital aspect of cell maintenance, especially for adherent cells like HEPG2. Over time, cells can become overconfluent, leading to changes in their behavior, morphology, and metabolic activity. Splitting cells prevents overcrowding, ensuring they have adequate space and nutrients to grow and function optimally. It also allows for the removal of dead or dying cells, ensuring a healthy culture.

## Preparations

1. Turn on the fan and light of the biosafety hood and open.
2. Spray your gloves with ethanol and allow them to air dry them.
3. Spray the surface of the hood with ethanol and wipe it down with a paper towel.
4. Get pre-aliquoted trypsin (1.5 mL) from the freezer and place under the hood.
5. Get a waste beaker and spray with alcohol; enough so that there is still some pooled at the bottom, and place under the hood.
6. Place EMEM media from the fridge under the hood.
7. Take 2 new T-25 flasks and place them under the hood. Label them with the passage number, the cell type & media type, the split ratio (1:8 or 1:80), the date, & initials.
8. Grab 2 sleeved 10mL pipettes and place under the hood.
9. Put T-25 flask with 70% confluent sample under the hood.

## Procedure

1. Loosen caps so they can be easily removed and ensure T-flasks are facing the correct direction.

* If trypsin is still frozen, thaw between hands before continuing.

2. Un-sleeve a 10mL pipette.
3. Take 10mL of EMEM and dispense 5mL into the 1:80 flask and 5mL into the 1:8 flask.
4. With the same pipette, take 6.5mL of EMEM and spray into the 1:8 flask.
5. Use the same pipette to remove the old media from the flask with the sample and discard it into the waste beaker.
6. Take all the pre-aliquoted trypsin and dispense into the flask with the sample.
7. Close the cap for trypsinized cells tightly.

* Incubate the cells with the trypsin for 5-10 minutes to allow them to split.
* Check the cells periodically under the microscope to make sure they are ready for the next step. They should no longer be adhered to the flask and should be floating as just a few cells, not big clumps of cells.

8. Once ready, return the cells under the hood and un-sleeve the second 10mL pipette.
9. Take the extra 6.5mL media from the 1:8 flask and dispense into the cell flask.
10. Suction up the cells, trypsin, and media from the flask, and dispense a few times to disperse any remaining chunks of cells.
11. Take 1mL of this mixture and disperse into the 1:8 flask.
12. Suction and dispense like the previous step to mix.
13. Take .5mL from the 1:8 flask and then dispense it into the 1:80 flask.
14. Suction and dispense to evenly mix.
15. Put flasks in the incubator and clean up.

## DNA Isolation

DNA extraction involves utilizing various reagents and centrifugation to isolate pure DNA. A specialized DNA extraction kit, equipped with the necessary washes and buffers, can be employed for this purpose. For our experiments, DNA isolation is essential since both GFP DNA, and V2 DNA are regularly used. Bacteria that carry these genes are cultivated and incubated, and the extracted DNA is then introduced to cells via transfection.

## Day 1: Petri Dish Setup

1. Get bacterial cells from the freezer.
2. Label an appropriate petri dish with cells (V2, GFP, etc.), date, and initials.
3. In the sterile area of a Bunsen burner, use a sterilized inoculation loop to streak the petri dish with bacterial cells.
4. Incubate at 37 °C for 24 hours.

## Day 2: Bacterial Transfer

1. Obtain a petri dish from the incubator.
2. Prepare a 250mL baﬄed Erlenmeyer ﬂask of 50mL of LB growth medium and label it with cells (V2, GFP, etc.), antibiotics, date, and initials.
3. Add 50uL of appropriate antibiotic to the ﬂask.
4. In the sterile area of a Bunsen burner, use a sterilized inoculation loop to add bacterial cells to the ﬂask.
5. Incubate the baﬄed Erlenmeyer ﬂask in a shaker at 37 °C and 300 RPM for 19-20 hours.

## Day 3: Isolation

1. Pour liquid from the Erlenmeyer flask into two 50mL tubes.
2. Centrifuge at 3000 RPM for 10 minutes.
3. Pour out liquid and upturn tubes onto a paper towel for 1 minute.
4. Resuspend each cell pellet with 600 µL of P1 Buffer.
5. Label 3 microcentrifuge tubes with cells (V2, GFP, etc.), date, and initials.
6. Split mixture into three microcentrifuge tubes (200 µL each).
7. Add 200 µL of P2 Buffer into the microcentrifuge tube.
8. Add 400 µL of P3 Buffer into the microcentrifuge tube.
9. Mix by inversion until all the pink color has disappeared.
10. Centrifuge 15,000 x g for 5 minutes (more if needed).
11. Remove supernatant (clear yellow liquid) from the microcentrifuge tube without disturbing the cell pellet and put it into a cartridge with a collection tube.
12. Centrifuge 15,000 x g for 1 minute.
13. Dump excess liquid from the collection tube.
14. Add 200 µL of DNA wash to the cartridge.
15. Centrifuge 15,000 x g for 30 seconds.
16. Dump excess liquid from the collection tube.
17. Repeat steps 14-16.
18. Add 200 µL of endo-wash to the cartridge.
19. Centrifuge 15,000 x g for 30 seconds.
20. Dump excess liquid from the collection tube.
21. Repeat 18-20 3X, I.E., 600ul total.
22. Dry centrifuge at 15,000 x g for 30 seconds without added wash.
23. Warm 150 µL DNA elution to 65 C.
24. Add 33 µL of DNA elution to the cartridge and place cartridge inside a microcentrifuge tube labeled with DNA, date, initials, and A (for first dilution).
25. Centrifuge at 15,000 x g for 2 minutes.
26. Repeat steps 24-27 two more times labeling "B" for the second dilution and "C" for the third dilution.
27. Measure each DNA absorbance with a nano dropper.
28. Label tubes with absorbance and ng/µL.
29. Freeze in the cell lab.

## Transfection

Transfection is a technique used to introduce foreign DNA or RNA into cells. This technique allows for the manipulation of cellular genetics, leading to the expression or inhibition of specific proteins or functions. In the context of this project, both GFP and V2 are transfected into cells. GFP serves as a control in the luciferase assay due to its ability to emit green fluorescence when viewed under a fluorescent microscope, and V2 is the experimental vector transfected into DNA to be evaluated in the luciferase assay.

## Day 1: 96-Well Plate Set Up

1. Remove old media from the pre-setup flask that is about 70% confluent.
2. Add 1.5mL trypsin to the flask.
3. Incubate for about 5-10 minutes, or until the cells are split.
4. Add 6.5mL fresh media to neutralize the trypsin.
5. Resuspend until it is a single cell suspension.
6. Count cells using a hemocytometer.
7. Add 10µL of cells from the flask to hemocytometer.
8. Count the four corner squares.
9. Average the counts of the four corner squares and multiply by 10,000.
10. Get cell stock volume by calculating:
    `(Avg Cell Count x 10,000) cells/mL = 30,000 cells`
11. Prepare 96-well plate:

Label with HEPG2, the date, and initials.
Add media and cell stock according to their count so that:
`µL cell stock + µL media = 100µL`

Incubate 96-well plate to allow cells to attach to the plate and grow. (24-48 hours)

## Day 2: Transfection

1. Prepare two microcentrifuge tubes.
2. Label one tube with D to use for the DNA solution.
3. Label one tube with L to use for the Lipofectamine 2000 solution.
4. Add Xµg\* (calculated using ng/mL labeled on DNA) of DNA to 25uL of Opti-MEM media into the D tube.
5. Mix DNA solution by inversion three times.
6. Vortex Lipofectamine 2000 for 30sec.
7. Add YµL\* of Lipofectamine 2000 to 25uL of Opti-MEM in the L tube.
8. Vortex solution of tube L for 30 sec.
9. Let solutions in both tubes incubate for 5 minutes at room temperature.
10. Remove old media from well in the prepared 96-well plate with 70% HEPG2 cell confluency.
11. Add 100uL of fresh MEM media to the well.
12. Add 50µL of the total solution of DNA and Lipofectamine 2000 to the well.
13. Pipette the total well volume up and down one time to mix.
14. Place the 96-well plate in the incubator for 24 hours.

\* X & Y are placeholders until the transfection optimization experiment is completed.

## Transfection Optimization Experiment

A transfection optimization experiment is a systematic approach to improve the efficiency of introducing foreign DNA into cells. By testing different concentrations of DNA and reagents, the optimal conditions for maximum transfection efficiency can be identified. Low transfection efficiency can hinder the success of experiments, leading to inconclusive results and wasted resources. By optimizing the transfection process, it ensures a higher proportion of cells receive genetic material.

1. Prepare 10 microcentrifuge tubes.

1. Label five tubes G1, G2, G3, G4, and G5 for the GFP DNA solution.
2. Label five tubes L1, L2, L3, L4, and L5 for the Lipofectamine 2000 solution.

2. Add 25uL x 5 (number of rows) of Opti-MEM to each of the tubes labeled G1 – G5.
3. Add 25ul x 5 (number of columns) of Opti-MEM to each of the tubes labeled L1 – L5.
4. Distribute GFP DNA to the (G) tubes in the following order:

1. 0.5uL to G1
2. 0.9uL to G2
3. 1.8uL to G3
4. 3.6uL to G4
5. 7.2uL to G5

5. Mix DNA solutions by inversion three times.
6. Vortex Lipofectamine 2000 for 30 seconds.
7. Add Lipofectamine 2000 to the (L) tubes in the following order:

1. 0.25ul to L1
2. 0.5uL to L2
3. 1.0 to L3
4. 2.0 to L4
5. 3.0 to L5

8. Vortex L-tubes for 30 seconds.
9. Let both solutions incubate for 5 minutes at room temperature.
10. While the solutions incubate, label 25 more microcentrifuge tubes for their corresponding plate location.

1. For row A, label five tubes A1 – A5.
2. For row B, label five tubes B1 – B5.
3. For row C, label five tubes C1 – C5.
4. For row D, label five tubes D1 – D5.
5. For row E, label five tubes E1 – E5.

11. Once ready, distribute the solutions of tubes G1 – G5 to their corresponding columns:

1. For G1, transfer 25uL to A1, 25uL to B1, etc.
2. For G2, transfer 25uL to A2, 25uL to B2, etc.
3. Continue this process for the rest of the remaining GFP DNA solutions and their columns.

12. Distribute the solutions of the tubes L1 – L5 in the corresponding rows:

1. For L1, transfer 25uL to A1, 25uL to A2, etc.
2. For L2, transfer 25uL to B1, 25uL to B2, etc.
3. Continue this process for the rest of the remaining Lipofectamine 2000 solutions and their rows.

13. Let the 25 solutions of DNA and Lipofectamine 2000 incubate for 15 minutes. While waiting, continue to the next step.
14. Remove old media from the wells in the prepared 96-well plate.
15. Add 100uL of fresh MEM media to each well.
16. Once incubation is complete, add 50uL of the mixed solution to its corresponding well.
17. Pipette the total volume of the well up and down once to mix.
18. Place the 96-well plate in the incubator for 24 hours.
19. After 24 hours, replace the media and document fluorescence with a microscope. Let incubate for 24 hours.
20. After 48 hours total, change the media again and document fluorescence with a microscope. Return to the incubator.

## Dual Luciferase Assay

The purpose of this assay is to test whether the V2 circuit detects cholesterol. The luciferase is a reporter gene that determines if the promoter is on or off. The luciferase gene is located inside the circuit, so when the substrate is added to the cells, it creates a reaction. This reaction produces luminescence, which is measured. The luminescence begins to decay after 12 minutes, so it is important to get luminescence readings before then. Low transfection efficiency will affect the results of the assay, so it is important to have multiple control wells to determine what or if anything went wrong in the assay. The ratio of the firefly to Renilla luminescence is compared between the control and transfected wells to determine if the V2 circuit and the assay are successful. Values of luminescence for firefly of the transfected cells should expect to be >10,000.

**Must be conducted a minimum of 48 hours after the previous transfection step**

**Make sure reagents are not older than 6 months.**

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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### Transfection Optimization

A transfection optimization experiment is a systematic approach to improve the eﬃciency of introducing foreign DNA into cells. By testing diﬀerent concentrations of DNA and reagents, the optimal conditions for maximum transfection eﬃciency can be identiﬁed. Low transfection eﬃciency can hinder the success of experiments, leading to inconclusive results and wasted resources. By optimizing the transfection process, it ensures a higher proportion of cells receive genetic material. To test whether the quantities or ratios of DNA or Lipofectamine 2000 need adjustment, or if reagents need replacement, 25 separate wells of a 96-well plate were rested with different volumes and ratios on HEPG2 cells in a grid. To verify efficiency of the transfection, the same grid was set up using C2C12 cells on the other side of the plate with the same pattern and ratios. The HEPG2 and C2C12 cells were transfected with increasing amounts of GFP DNA: 0.5uL, 0.9uL, 1.8uL, 3.6uL, 7.2uL. The HEPG2 and C2C12 cells were also transfected with increasing amounts of Lipofectamine 2000: 0.5uL, 1.0uL, 2.0uL, 3.0uL.

![Transfection Optimization of HEPG2 Cells](https://static.igem.wiki/teams/4896/wiki/image5.png)

Figure 1. Transfection Optimization of HEPG2 Cells

![Transfection Optimization of C2C12 Cells](https://static.igem.wiki/teams/4896/wiki/image3.png)

Figure 2. Transfection Optimization of C2C12 Cells

The results showed that the transfection was overall successful for both the HEPG2 and C2C12 cells. The optimal ratio of DNA to Lipofectamine was found to be 1.8uL to 1.0uL for HEPG2 cells.

### Dual Luciferase Assay

The purpose of this assay is to test whether the V2 circuit detects cholesterol. The luciferase is a reporter gene that determines if the promoter is on or oﬀ. The luciferase gene is located inside the circuit, so when the substrate is added to the cells, it creates a reaction. This reaction produces luminescence, which is measured. The luminescence begins to decay after 12 minutes, so it is important to get luminescence readings before then. Low transfection eﬃciency will aﬀect the results of the assay, so it is important to have multiple control wells to determine what or if anything went wrong in the assay. The ratio of the ﬁreﬂy to Renilla luminescence is compared between the control and transfected wells to determine if the V2 circuit and the assay are successful. Values of luminescence for ﬁreﬂy of the transfected cells should expect to be >10,000.

![Dual Luciferase Assay of Vector V2 at Low, Standard, and High CHolesterol Conditions](https://static.igem.wiki/teams/4896/wiki/image6.png)

Figure 3. Dual Luciferase Assay of Vector V2 at Low, Standard, and High CHolesterol Conditions

The dual luciferase assay in Figure 3 did not provide significant results. However, it did allow us to characterize the SRE promoter as sensitive to cholesterol. These preliminary results show that the promoter is active at low cholesterol levels and inactive at high cholesterol levels.

![Dual Luciferase Assay of Vector](https://static.igem.wiki/teams/4896/wiki/image2.png)

Figure 4. Dual Luciferase Assay of Vector V2

The firefly and renilla genes in the V2 vector were shown to be functioning.

![Dual Luciferase Assay of Putative Stable Cells Lines](https://static.igem.wiki/teams/4896/wiki/stable-cell-lines-dual-luciferase-assay-w-error-bars.jpg)

Figure 5. Dual Luciferase Assay of Putative Stable Cells Lines

Three putative stable cell lines were transfected with V2 vector DNA and then assayed. Cell lines exhibited signals from the renilla luciferase but not the firefly luciferase. This would imply that the V2 vector was only partially transfected into the cells such that the constitutive promoter was transfected while the pSRE inducible promoter was not. Further screening of cell lines transfected with the V2 vector is required.

### qPCR

The goal is to confirm the gene targets that, in cells, may be employed to prevent the formation of cholesterol and serve as the cornerstone for the creation of the V3 gene circuit. There are 17 enzymes that work together to catalyze the cholesterol production pathway in liver cells. We select the enzymes squalene monooxygenase (SQLE) and farnesyl-diphosphate farnesyltransferase 1 (FDFT1) based on previous research. In order to determine which of these genes are potential targets for the inhibitory mechanism of the gene and to assess the gene expression of these two enzymes, it was necessary to analyze the levels of expression of the genes encoding FDFT1 and SQLE in wild-type HEPG2 cells.

![SQLE Average ΔCq Values](https://static.igem.wiki/teams/4896/wiki/image1.png)

Figure 6. SQLE Average ΔCq Values

![FDFT1 Average ΔCq Values](https://static.igem.wiki/teams/4896/wiki/image4.png)

Figure 7. FDFT1 Average ΔCq Values

\* indicates significance with a p-value < 0.05

Lower ΔCq values indicate higher gene expression, with GAPDH serving as a control gene. Negative ΔCq values represent reduced gene expression. The results showed that SQLE siRNA successfully reduced SQLE gene expression, as its ΔCq values were significantly lower than controls. However, FDFT1 siRNA did not effectively reduce FDFT1 gene expression, as its ΔCq values were less negative than controls. This suggests that the siRNA, not the delivery method (RNAiMAX Lipofectamine), caused the gene inhibition. Future research will involve integrating the SQLE siRNA mechanism into a V3 circuit to regulate cholesterol synthesis and exploring additional siRNAs for FDFT1 or other cholesterol synthesis genes for potential inclusion in the V3 circuit.

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