Composite

Part:BBa_K5441012

Designed by: Lok Yin Wong   Group: iGEM24_PLKLFC   (2024-10-01)

PSMA_Gluc

High accuracy prostate cancer detection system with PSMA promoter

PSMA promoter with Gluc at its downstream.

Evaluation of detection ability theoretically

PSMA (prostate-specific membrane antigen) is a type II transmembrane glycoprotein consisting of 750 amino acids, encoded by the folate hydrolase 1 gene, which is situated on the short arm of chromosome 11. [1]

Histologically, PSMA is present at low levels in the epithelium of benign prostate tissue but shows a significant increase in expression—by 100 to 1,000 times—in prostate adenocarcinomas [2][3]. It is found in the majority of tumours (90-100%)[7], and higher levels of PSMA expression have been positively correlated with several indicators of tumour aggressiveness, such as Gleason grade[3], tumour stage[4], biochemical recurrence[5], and castration resistance[6].

Thus, PSMA has been proven as an effective biomarker for prostate cancer (PCa). Moreover, it shows fewer false-positive results than the commonly used PSA[8].

To produce PSMA, PCa cells contain substances that activate the promoter in its copy of the organism gene, which allows the transcription of PSMA to be facilitated. The same substance activates the PSMA promoter in this part and transcribes Gaussia Luciferase (Gluc) to detect cancer cells. Gluc is a protein which is naturally secreted into cell media (in vitro) and urine (in vivo), allowing for easy and safe quantification without harming the patient’s normal cells.

Evaluation of detection ability experimentally

In our project, we transfected different concentrations of plasmids containing this composite part into various concentrations of the PSMA-positive cell line. (MLLB-2)

To quantify the luminescence, a Gaussia Luciferase Flash Assay is performed. In the reaction, Coelenterazine (substrate) is oxidised by Gluc (enzyme) to produce light with an emission maximum of 485nm. 20μL Cell media is first extracted from the culture, followed by the addition of 50μL Coelenterazine working solution. Luminescence at all wavelengths is then measured with a luminometer (multimode plate reader) immediately.

We studied if the concentration of the plasmid, that of MLLB-2 cells, or the combination of both will affect the luminescence signals. Our experiment includes setups with plasmids at 9.11e-4 μg/uL, 4.55e-3 μg/uL, and 9.09e-3 μg/uL. Three cancer cell concentrations, at 10,000, 50,000, and 100,000 cells per 0.33 mm2, were included for each plasmid concentration. A control with no transfected plasmids is included. Using two-way ANOVA, we obtained the following results: 

- Individual cancer cell concentration: p < 0.001 
- Individual plasmid concentration: p = 0.036 < 0.05 
- A combination of both (plasmid conc. * cell conc.): p = 0.006 < 0.05

Our results demonstrated that both variables affect the bioluminescence given out by Gluc.

Fig. 1

Fig. 2

Fig. 1: The setups without plasmids, among all cancer cell concentrations, do not show any significant differences in luminescence readings. This indicates that our engineered plasmid does not induce elevated or reduced luminescence levels in cells. In a low plasmid concentration, high and low concentrations of cancer cells display significant differences (p = 0.002 < 0.01), which shows that highly concentrated cancer cells have a significantly lower luminescence value (12.95 RLU) than that of low-concentration cancer cells (29.94 RLU). In medium and high plasmid concentrations, low-concentration cancer cells always have significantly higher luminescence values than the ones found in medium and high concentrations (p < 0.01).

Fig. 2: A more obvious result is observed, in which a high concentration of plasmids combined with any concentrations of cancer cells results in a low luminescence value. We conclude that to detect cancer cells effectively, one should use the following plasmid-to-cell ratios: 4.55 μg/mL plasmids:10,000 cells 0.911μg/mL plasmids:50,000 cells In general, low levels of cancer cell concentrations require high levels of plasmid concentrations to maximize the luminescence. Vice versa, higher levels of cancer cell concentrations require lower levels of plasmid concentration for a maximum luminescence value. This graph can be used for further extrapolation upon the availability of more evidence.

High concentrations of prostate cancer cells produce a larger amount of acidic metabolic waste in a higher rate compared to that of both low and high concentrations of prostate cancer cells, the more acidic culture medium of high concentration prostate cancer cells lowers the rate of their protein synthesis.

Advantages for our system

This design utilises the same system of PSMA_GFP (Part BBa_K5441010) and has the same advantages as it. By using this promoter + reporter gene design, theoretically, this system can detect almost all kinds of diseases. As long as researchers find a suitable biomarker for the disease, a promoter in the genome can be isolated and used in all kinds of systems. Furthermore, the reporter gene used is a naturally secreted Gaussia Luciferase, which allows its level in the blood or urine to be used as a marker to monitor different in vivo biological events such as tumor growth and response to therapy, while not being invasive to patients themselves. [9] In the future, we hope that this system can be customised to every researcher’s needs, bringing convenient disease detection to allow for early cure.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1839
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 630


[1] Rinker-Schaeffer CW, Hawkins AL, Su SL, et al. Localization and physical mapping of the prostate-specific membrane antigen (PSM) gene to human chromosome 11. Genomics. 1995;30:105–108.

[2] Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C. Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res. 1997;3:81–85.

[3] Bostwick DG, Pacelli A, Blute M, Roche P, Murphy GP. Prostate specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma. Cancer. 1998;82:2256–2261.

[4] Perner S, Hofer MD, Kim R, et al. Prostate-specific membrane antigen expression as a predictor of prostate cancer progression. Hum Pathol. 2007;38:696–701.

[5] Ross JS, Sheehan CE, Fisher HAG, et al. Correlation of primary tumor prostate-specific membrane antigen expression with disease recurrence in prostate cancer. Clin Cancer Res. 2003;9:6357–6362.

[6] Wright GL, Grob BM, Haley C, et al. Upregulation of prostate-specific membrane antigen after androgen-deprivation therapy. Urology. 1996;48:326–334.

[7] Ceci, F., & Fanti, S. (2019). PSMA-PET/CT imaging in prostate cancer: why and when. Clinical and Translational Imaging, 7(6), 377–379. https://doi.org/10.1007/s40336-019-00348-x

[8] Velonas, V., Woo, H., Remedios, C., & Assinder, S. (2013). Current status of biomarkers for prostate cancer. International Journal of Molecular Sciences, 14(6), 11034–11060. https://doi.org/10.3390/ijms140611034

[9] Wurdinger T. A secreted luciferase for ex vivo monitoring of in vivo processes. Nat Methods. 2008;5:171–3.

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