-Experimental Design-

Principle of our project

1. Detection of prostate cancer

The plasmid will be transported to prostate cancer cells and Gluc, a fluorescence protein, will be expressed by the plasmid in the cancer cells and be transmitted to urine. The intensity of bright blue light emitted by the sample will be measured by plate reader and thus prostate cancer will be detected.

2. Killing of prostate cancer cells

Other than Gluc, Bax is another protein that will be expressed by another plasmid we synthesise. Bax can trigger apoptosis of prostate cancer cells, enabling us to kill the cancer cells as soon as possible.

Our experimental design is divided into three parts: construction of plasmid, cell line as well as the result verification.

1. The Cell Line

Aim:

To culture cells for verification of our plasmid design and concept.

Cell lines used:

According to Bakht et al. (2019) neuroendocrine prostate cancer cells do not have PSMA expression. Therefore, we have chosen PNEC 30 as the PSMA-negative prostate cancer cell line. Moreover, for MLLB-2, we have found that it has been used as PSMA-positive prostate cancer cells from the website of American Type Culture Collection (ATCC). Therefore, we use MLLB-2 as a PSMA-positive prostate cancer cell line.

Purpose of each cell line:

MLLB-2 and PNEC30 are experimental cell lines with prostate cancer, MLLB-2 ensures that the plasmid can be expressed under an environment with high PSMA level, while PNEC30 ensures that the PSMA promoter will not be activated under an environment without any PSMA.

Experiment:

Our cell line is used in the experiment in which our plasmids are added to the cells. Moreover, we will be using MTT cell assay to measure the natural growth rate of the prostate cancer cells, which can act as a control of the experiment.

Cell lines used:

1. MLLB-2
2. PNEC30

With MLLB-2, it allows us to study the activity of our plasmid towards PSMA-positive prostate cancer cells, an increase in light intensity of cells under plate reader indicates a successful detection of PSMA-positive prostate cancer cells, while a decrease in cell concentration indicates a successful killing of them.

With PNEC30, it allows us to study the activity of one of our plasmids (pENTR1A-PSMA-GFP ) towards PSMA-negative prostate cancer cells, no fluorescence signal detected indicates that PSMA is an effective biomarker for identifying and detecting prostate cancer detect and even kill PSMA-negative prostate cancer cells.

2. Construction of Plasmid

Figure 1: The designs of our plasmid

Plasmid construction

The plasmid serves as a crucial role in locating the cancer cells as well as expressing the protein in the cancer cells. We have chosen several genes that will be put into the plasmid, and they are probasin promoter (PB promoter), PSMA promoter, the gene that will express gaussia luciferase (Gluc), as well as the gene that will express BAX. As we will be using a probasin promoter as the second gate in our AND-GATE system, and to activate the expression of gene, we have chosen a plasmid that does not contain any promoter, which is pENTR1A-NTAP-A (w322-1) from Addgene.

However, at the start of the experiment, we will first choose PSMA promoter as our promoter and GFP as the reporter gene, as GFP is commonly found and we can verify the sequence of PSMA promoter through constructing a plasmid with PSMA-GFP gene. Then, we can verify the sequence of gene that expresses Gluc by replacing the gene of expressing GFP. After that, we can verify the sequence of the probasin promoter by replacing the PSMA promoter. At last, we can verify the sequence of BAX by adding BAX to the constructed plasmid.

Figure 2: pENTR1A-NTAP-A (w322-1) (Source: Addgene)

However, as we are finding the sequence of the genes from GenBank, we will verify the sequence of genes of each component by performing experiments after constructing the plasmids.

Go to the description page to learn more about the genes we put into the plasmid.

Experiment 1:

Aim:

To construct a plasmid with PSMA-GFP gene and verify the sequence of PSMA promoter.

Constructs:

pENTR1A-NTAP-A (w322-1) with PSMA-GFP

Figure 3: Map for PSMA_GFP gene

Strain:

E.coli DH 5 alpha

Methods:

The plasmid pENTR1A-PSMA-GFP is constructed, and colony PCR has taken place to ensure the gene of interest, PSMA-GFP, has been implanted into the plasmid. After the plasmid has been constructed, we will transfect the plasmid to the cell line to ensure that the PSMA promoter will only be activated when PSMA is present in the environment. It can be tested by checking whether a green light with wavelength of 510 nm is detected, which indicates the presence of GFP (Green Fluorescent Protein).

Experiment 2:

Aim:

To construct a plasmid with PSMA-Gluc gene and verify the sequence of gene that express Gluc.

Constructs:

pENTR1A-NTAP-A (w322-1) with PSMA-Gluc

Figure 4: Map for PSMA_Gluc gene

Strain:

E.coli DH 5 alpha

Methods:

The plasmid pENTR1A-PSMA-Gluc is constructed, and colony PCR has taken place to ensure the gene of interest, PSMA-Gluc, has been implanted into the plasmid. After the plasmid has been constructed, we will transfect the plasmid to the cell line to ensure that Gluc can be expressed from the plasmid when the promoter is activated, which can be checked by detecting the presence of a bright blue light with wavelength of 480 nm.

Experiment 3:

Aim:

To construct a plasmid with Pb-Gluc gene and verify the sequence of probasin promoter (Pb).

Constructs:

pENTR1A-NTAP-A (w322-1) with Pb-Gluc

Figure 5: Map for Pb_Gluc gene

Strain:

E.coli DH 5 alpha

Methods:

The plasmid pENTR1A-Pb-Gluc is constructed, and colony PCR has taken place to ensure the gene of interest, Pb-Gluc, has been implanted into the plasmid. After the plasmid has been constructed, we will transfect the plasmid to the cell line to ensure that the Probasin promoter will only be activated at the prostate gland. It can be tested by checking whether a bright blue light with wavelength of 480 nm is detected, which indicates the presence of Gluc.

Experiment 4:

Aim:

To construct a plasmid with Pb-Gluc-BAX gene and verify the sequence of gene that express BAX.

Constructs:

pENTR1A-NTAP-A (w322-1) with Pb-Gluc-BAX

Figure 6: Map for Pb_Gluc_BAX gene

Strain:

E.coli DH 5 alpha

Methods:

The plasmid pENTR1A-Pb-Gluc-BAX is constructed, and colony PCR has taken place to ensure the gene of interest, Pb-Gluc-BAX, has been implanted into the plasmid. After the plasmid has been constructed, we will transfect the plasmid to the cell line to ensure that BAX can be expressed from the plasmid when the promoter is activated, which can be checked by measuring the reduction in the density of cancer cells.

Figure 7: A graphics to show the final product

3. Result Verification

Result verification is an important part of our project. We will collect data from our experiment and conduct the modelling part of our project. Also, this part allows us to know whether we have invented our medication successfully.

In the previous parts, we have constructed a plasmid. In order to check whether prostate cancer cells can be detected and killed after constructing it, we will conduct the following experiments.

Experiment 1:

Aim:

To test whether our medication can detect PSMA-positive prostate cancer cells.

Methods:

1. Inject the medication (plasmid pENTR1A-PSMA-Gluc and plasmid pENTR1A-Pb-Gluc) into the different combinations of cell line and collect the sample for inspection.
2. Measure the light intensity of the sample with a plate reader and collect the data.

Figure 8: A graphics to show the step in experiment 1

Experiment 2:

Aim:

To test whether our medication can kill prostate cancer cells.

Methods:

1. Inject the medication (plasmid pENTR1A-Pb-Gluc-BAX) into different combinations of cell line.
2. Observe the cell line under a microscope. Check whether the density of living cancer cells decreases.

Figure 9: A graphics to show the step in experiment 2

4. Contingency Plan

We have made a contingency plan for our project, so that we can continue our experiment even if some of the above parts are not done successfully.

Experiment:

Aim:

To ensure the plasmid can be expressed under high PSMA level even if we fail to subculture the cell line.

Strain:

E.coli BL21

Methods:

1. Introduce our plasmid (pENTR1A-PSMA-Gluc) into E.coli BL21 by bacteria transformation.
2. Add the transformed E.coli BL21 to different PSMA concentrations, which the PSMA concentrations represent the average serum PSMA value of real patient, proposed by Xiao et al. (2001)

Groups	Average serum PSMA value (ng/mL)
Extreme value	1,000
Prostate cancer patients	623.1
Normal men whose age >50	359.4
Normal men whose age <50	272.9
Benign prostate hyperplasia (BPH) patients	117.1
Control	0

Table 1:Average serum PSMA value of real patients

3. Measure the light intensity of the bright blue light emitted by gaussia luciferase with a plate reader.

Reference

• [1] Bakht, M. K., Derecichei, I., Li, Y., Ferraiuolo, R., Dunning, M., Oh, S. W., Hussein, A., Youn, H., Stringer, K. F., Jeong, C. W., Cheon, G. J., Kwak, C., Kang, K. W., Lamb, A. D., Wang, Y., Dong, X., & Porter, L. A. (2019). Neuroendocrine differentiation of prostate cancer leads to PSMA suppression. Endocrine Related Cancer, 26(2), 131–146. https://doi.org/10.1530/erc-18-0226
• [2] Xiao, Z., Adam, B., Cazares, L. H., Clements, M. A., Davis, J. W., Schellhammer, P. F., Dalmasso, E. A., & Wright, G. L. (2001). Quantitation of serum prostate-specific membrane antigen by a novel protein biochip immunoassay discriminates benign from malignant prostate disease. PubMed, 61(16), 6029–6033. https://pubmed.ncbi.nlm.nih.gov/11507047