{% extends "layout.html" %} {% block title %}Results{% endblock %} {% block lead %}{% endblock %} {% block image %}https://static.igem.wiki/teams/5441/homepage/dsc00165.png{% endblock %} {% block page_content %}

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

Fig 1. Plasmid map of pENTR1A-PSMA-GFP

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

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

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

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.

(a) Successful Cloning of pENTR1A-PSMA-Gluc

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

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

  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,

In Figure 9,

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.

(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

Conclusion

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

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