As a result of our work, we have provided detailed experimental findings related to our design. We employed a divide-and-conquer strategy, testing each component individually through wet experiments to confirm their functionality. There are three results showing the success of our engineering cycle:

(1) Testing PSMA Promoter

Objective: Our first composite part (PSMA-GFP, BBa_K5441010) is used to confirm that the PSMA promoter gene is a viable option for detecting the presence of PSMA in prostate cancer (PCa) cells. This is crucial as we selected PSMA as our biomarker for our project. Our experimental design can be reviewed at the Design Page.

(a) Successful Cloning of pENTR1A-PSMA-GFP

• Due to its convenient detection of quantification, GFP (Green Fluorescent Protein) was chosen as the reporter gene to verify PSMA promoter’s function.

Fig 1. Plasmid map of pENTR1A-PSMA-GFP

• We constructed the plasmid pENTR1A-PSMA-GFP by ligating parts of the pENTR1A vector with our PSMA-GFP gene. In order to verify whether the gene was ligated, after transformation, we extracted the plasmids which was followed by restriction digestion. To confirm its size, only one restriction enzyme was used (BamHI) to linearise the gene. The length of the gene is confirmed using gel electrophoresis shown in figure 2.

Fig 2. Digested pENTR1A-PSMA-GFP and pENTR1A-PSMA-Gluc

(b) Successful in Function Verification of pENTR1A-PSMA-GFP

• pENTR1A-PSMA-GFP was then transfected into a PSMA-positive PCa cell line (MLLB-2), and another PSMA-negative PCa cell line (PNEC30) as a negative control. Another negative control setup was included with only PSMA-positive PCa cells but not any plasmids.

• A Nikon Eclipse Ti inverted microscope, along with a blue light source, was used to visualize the fluorescence given out inside the cells. Fluorescence was only observed in set-ups with both MLLB-2 and pENTR1A-PSMA-GFP added. Others did not show any fluorescence.

• Fluorescence signal emitted from GFP was quantified using a multimode microplate reader,
• using an excitation wavelength of 470 nm and emission wavelength of 510 nm.

• The results of the fluorescence signal are shown in the figures below.

Fig 3a. MLLB-2 (PSMA-positive) with PSMA-GFP transfected with GFP filter	Fig 3b. MLLB-2 (PSMA-positive) with PSMA-GFP transfected without filter

Fig 4a. MLLB-2 (PSMA-positive) without PSMA-GFP transfected with GFP filter	Fig 4b. MLLB-2 (PSMA-positive) without PSMA-GFP transfected without filter

Fig 5a. PNEC30 (PSMA-negative) with PSMA-GFP transfected with GFP filter	Fig 5b. PNEC30 (PSMA-negative) with PSMA-GFP transfected without filter

(c) Quantification of the Fluorescence Signal of Different Concentrations of pENTR1A-PSMA-GFP on PSMA-positive PCa Cell Line

The results of said quantification graphs are shown below :

Fig 6a. GFP fluorescence at 0.2 μg / 60 μL plasmid	Fig 6b. GFP fluorescence at 0.4 μg / 60 μL plasmid	Fig 6c. GFP fluorescence at 0.8 μg / 60 μL plasmid

• Three control setups were used. Each setup contains a different concentration of PSMA-positive PCa cells (10,000 cells per 0.33 mm², 20,000 cells per 0.33 mm² and 40,000 cells per 0.33 mm²). These did not have any plasmids transfected.

• We then tested for the effect of PSMA-GFP towards PSMA-positive PCa cell lines by transfecting 3 different concentrations of pENTR1A-PSMA-GFP (0.00333 μg / μL, 0.00667 μg / μL, 0.0133 μg / μL) into 3 different concentrations of PSMA-positive PCa cells (same as that in the 3 control setups). All our setups demonstrate significant differences (p < 0.05, independent T-test) when compared to the controls.

• The only exception to this is the setup with the highest plasmid concentration (0.0133 μg / μL) and 20,000 cells per 0.33 mm². It showed no statistical difference when compared to the control.

Conclusion

The above result shows that PSMA promoter can be activated by PSMA-positive PCa cells, and lead to a high expression of the downstream gene. From the above engineering success, we could conclude that PSMA is an effective biomarker for prostate cancer and verify the function of the PSMA promoter. High concentration of plasmid (0.0133 μg /μL) may be possible for inhibiting the GFP expression.

(2) Testing Gaussia Luciferase Reporter Gene

Objective: Having already concluded that PSMA is an effective biomarker for prostate cancer from the first engineering cycle, we then moved to the second engineering cycle. However, GFP is toxic to the human body. As such, we replaced GFP with the Gaussia Luciferase (Gluc) gene in our construct, which means that the presence of secreted Gluc indicates a possible existence of prostate cancer cells in the patient’s body.

• In vivo, Gluc is naturally secreted outside cells and can be detected through the urine. Utilising this property of Gluc, in a clinical research context, when our plasmid is injected to the bloodstream, we can test whether the patient’s urine contains Gluc with a Gaussia Luciferase Flash/Glow Assay Kit. If a patient’s urine contains Gluc, it can be deduced that the patient’s cells are also producing high levels of PSMA, indicating the presence of prostate cancer. The test kit can be performed within thirty minutes, making this a faster and more economical pre-screening tool than a traditional PET scan.

• Easy detection employing Gluc is an essential part of our project. We validated the function of Gluc using our second composite part (PSMA-Gluc, BBa_K5441012). Our experimental design can be reviewed at Design.

(a) Successful Cloning of pENTR1A-PSMA-Gluc

• We constructed the plasmid pENTR1A-PSMA-Gluc by ligating parts of the pENTR1A vector with our PSMA-Gluc gene. In order to verify whether the gene was ligated, after transformation, we extracted the plasmids which was followed by restriction digestion. To confirm its size, only one restriction enzyme was used (BamHI) to linearise the gene. The plasmid map and the length of the gene is confirmed using gel electrophoresis shown in the figure below.

Fig 7. Digested pENTR1A-PSMA-GFP and pENTR1A-PSMA-Gluc

(b) Successful Quantification of Luminescence Signal Produced by Various Concentrations of pENTR1A-PSMA-Gluc in PSMA-positive PCa Cell Line

• pENTR1A-PSMA-Gluc was then transfected into a PSMA-positive PCa cell line (MLLB-2), and another PSMA-negative PCa cell line (PNEC30) as a negative control. Another negative control setup was included with only PSMA-positive PCa cells but not any plasmids.
• The results from the Gaussia Luciferase Flash Assay Kit were collected via the use of a luminometer (multimode microplate reader), choosing all wavelengths for luminescence. With the help of iGEM team HKUST 2024 & supervision under Dr. Yu from G.T. College, we were able to ascertain the results below.

Fig 8. Bar chart of Gluc luminescence at various cells and plasmid concentrations	Fig 9. Broken line chart of Gluc luminescence at various cells and plasmid concentrations

• Utilizing two-way ANOVA, we studied whether the plasmid, pENTR1A-PSMA-Gluc (BBa_K5441012), concentration, cancer cell (MLLB-2) concentration or a combination of both can affect the luminescence signal. Our results demonstrates the relationships of the following parameters with luminescence measured:

1. individual cancer cell concentration: p < 0.001
2. individual plasmid concentration: p = 0.036 < 0.05
3. combination of both (plasmid * cancer cell no#): p = 0.006 < 0.05

This result shows that both parameters contribute to the luminescence level.

In Figure 8,

• Our control setup does not contain any transfected plasmids. The wells without plasmids, no matter the number of cancer cells, shows no significant differences in luminescence reading. This proves that our engineered plasmid does not induce any elevated nor reduced levels of luminescence.

• In a low plasmid concentration (0.000911 μg / μL), both high-concentration (100,000 cells per 0.33 mm²) and low-concentration (10,000 cells per 0.33 mm²) cancer cells display significant differences (p = 0.002, < 0.01). This shows that high-concentration of cancer cells have a significantly lower luminescence value (12.95 RLU) than that of low-concentration cancer cells (29.94 RLU).

• In medium (0.00455 μg / μL) and high (0.00909 μg / μL) plasmid concentrations, low-concentration cancer cells always have a significantly higher luminescence value than those measured in medium and high-concentration of cancer cells (p < 0.01).

In Figure 9,

• A more obvious result can be seen. We found that a high concentration of plasmids combined with any cancer cell concentration leads to lower luminescence.

• In order to detect cancer cells effectively, one should employ the following plasmid-to-cells ratios:
• 4.55 μg/mL plasmids:10,000 cells
• 0.911μg/mL plasmids:50,000 cells

Conclusion and Prediction

In general, low-level cancer cell concentration requires high-level plasmid concentration to maximize the luminescence signal. On the contrary, high-level cancer cell concentration requires a low-level plasmid concentration.

However, the pattern that lower levels of plasmid concentration results in a lower level of luminescence is observed in all cancer cell concentrations.

According to our graphs, the trend shows that when a lower cell concentration is paired with a medium concentration of plasmid, the luminescence value observed is the highest.

At a high-concentration of cancer cells, it will produce the lowest amount of luminescence among all three concentrations of plasmids we studied. It can be explained by the fact that as high concentrations of prostate cancer cells produce larger amounts of acidic metabolic waste at a higher rate, the more acidic culture medium of high concentration prostate cancer cells lowers the rate of their protein synthesis.

Fig 10. Linear regression on optimum performance of Gluc luminescence at different cells and plasmid concentrations

This graph can be used for further extrapolation upon the availability of more evidence.

Fig 11. Prediction of the performance on Gluc luminescence at low cell concentration

(3) Testing the killing function of BAX

Objective: After the engineering success of Gluc, we moved on to the third stage of the engineering cycle. In addition to the detection of prostate cancer cells (MLLB-2) with Gluc, we aim to also kill the cancer cells. Thus, in our plasmid, we inserted the gene of an apoptosis regulator BAX, which promotes apoptosis of cells into our plasmid PB-Gluc, producing PB-Gluc-BAX. This PB promoter targets any prostate cancer cells. To test for the effect of killer gene BAX on prostate cancer cells, we seeded PSMA-positive prostate cancer cells into 36 wells, all containing the same cell concentration (30000 cell per 0.33 mm²), in a 96-well plate.

• The reactions between PB-Gluc-BAX and cancer can be monitored by MTT Cell Viability Assay and it can be measured by absorption using the wavelength 540 nm.

(a) Successful Cloning of pENTR1A-PSMA-Gluc-BAX

In order to verify whether the gene was ligated using mammalian plasmid, we utilized plasmid extraction, followed by restriction digestion. The diagram shown below are the plasmid map and the results of the linear plasmid under gel electrophoresis.

Fig 12. Digested pENTR1A-PB-Gluc-Bax

(b) Successful Function Verification of pENTR1A-PSMA-Gluc-BAX

The graphs below shows the absorption of the reaction between Pb-Gluc-BAX and cancer cells.

Fig 13. Broken line chart of absorbance by MTT cell assay under different plasmid concentrations at different days

Fig 14. Bar chart of absorbance by MTT cell assay under different plasmid concentrations

Fig 15. Bar chart of absorbance by MTT cell assay under different days

• The setup without pENTR1A-PB-Gluc-Bax demonstrates a faster growth rate on the second day than first (p < 0.001). This proves that our wells were suitable mediums for the cells to grow from days 0 to 2 of the experiment.

• In low (0.00124 μg / μL) and high (0.00496 μg / μL) concentrations of transfected plasmids, the rate of increase in cancer cells is significantly slower than the setups without plasmids transfected from day 0 to day 2 (p < 0.05). There is also a significant drop in the number of cancer cells in the setup with medium (0.00248 μg / μL) concentration of pENTR1A-PB-Gluc-BAX transfected.

Conclusion

• On day 3, in the setup with medium concentration (0.00248 μg / μL) of plasmids, cell numbers dropped more significantly than lower and higher concentrations of plasmids. We found that the setup with a medium concentration of plasmids had number of cancer cells dropped significantly sharper than day 0.

• From days 0 to 2, low and high concentrations of cancer cells increased significantly. It is however, still much lower than the increase found in the setup with no transfected plasmids.

• pENTR1A-PSMA-Gluc-BAX can significantly reduce the growth rate of PSMA-positive PCa Cell (MLLB-2) in the well. Moreover it can kill the PSMA-positive PCa Cell (MLLB-2) in the well at a right concentration of pENTR1A-PSMA-Gluc-BAX.

Day 0 (Initial)	Day 1	Day 2	Blank (With only culture medium and MTT assay reagents)
Fig 16. Real-time pictures of colour of cells in the wells after treated with medium concentrations of pENTR1A-PB-Gluc-BAX under MTT Cell Viability Assay