iGEM 2024 — Team plklfc

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## Lab Notebook

![](https://static.igem.wiki/teams/5441/notebook/marchh.png)
[![](https://static.igem.wiki/teams/5441/notebook/inspiration.png)](#Mar Week 1)
[![](https://static.igem.wiki/teams/5441/notebook/deliver.png)](#Mar Week 2)
[![](https://static.igem.wiki/teams/5441/notebook/guides.png)](#Mar Week 3)

![](https://static.igem.wiki/teams/5441/notebook/img-2584-1.png)

##### Week 1: Inspiration from other teams

* Eg. Tsinghua 2023, McGill 2023
* Combined **other teams’ ideas** with our previous ideas
* **Protein level** (antibody) → **Genetic level** (promoters and reporter genes)
* Tsinghua: AND gate system

+ using multiple promoters to increase specificity
+ Eg. PSA promoter + PSMA promoter, PSA promoter + AR promoter

* Tsinghua: PD-L1 nanobody

+ With the aid of immune system kills cancer cells

* McGill: induced pyroptosis of cancer cells
* PLKLFC 2023: PSMA has a high correlation with PCa

+ + AND gate is difficult to design
+ Decided to use PSMA promoter only
+ PSMA promoters should only be activated **inside cells**
+ Does not cause harm to nearby cells

![](https://static.igem.wiki/teams/5441/notebook/img-2585-1.png)

##### Week 2: Delivery of plasmid to prostate

* Aim: to make it as **convenient** and **painless** as possible
* Inject plasmids into the bloodstream by intravenous injection → how to make it travel to the prostate? 1
* Plasmids degrade in the blood 3

+ Need “carriers” to transport them
+ Idea: polymers (small molecules of plastics) to prevent degradation 5

* Potential polymer 1: Hyaluronic acid (found in the fluids in the eyes and joints) 8

+ Binds to CD44 (overexpressed at tumour) and releases plasmid inside 9
+ Problem: unspecific to PCa 10,11

- ✓ Use promoters inside the plasmid to confirm the location
- 👎🏻 Decrease in [plasmid] before reaching prostate

* Potential polymer 2: Chitosan - sugar from the outer skeleton of shellfish
* Potential polymer 3: PAMAM (Poly(amidoamine) dendrimers) - plastics

+ Utilised in previous research regarding gene therapy for prostate cancer (Tai et al., 2020)
+ Problem: cytotoxic to cells 17
+ Solution: Attach polyethylene glycol (PEG) to reduce cytotoxicity (Guo, X., Wang, 2017) 20

![](https://static.igem.wiki/teams/5441/notebook/img-2589-1.png)

##### Week 3: Guides of polymers - Aptamers

* How to target prostate with the polymer? - Aptamers1

+ Sections of DNA/RNA/peptides that target specific molecules 3,4
+ High specificity 5,6
+ Produced in a batch → reduces production costs
+ Reversible denaturation (unlike antibodies)
+ Less immunogenic than antibodies
+ Can be attached to nanoparticles/polymers

* Potential aptamer 1: EpDT3 16

+ Binds to EpCAM (epithelial cell adhesion molecule, overexpressed in some tumour cells) 17
+ 👍🏻EpCAM is highly expressed in metastasized PCa cells → detect later stages of cancer 19
+ 👎🏻Still non-specific to PCa

* Dual aptamer design 23

+ A10-3.2: PSMA +ve (but only 80% of PCa patients have PSMA)
+ DUP-1: PSMA -ve
+ Used in a lot of research previously
+ We can add a constitutive promoter inside our plasmid to express the reporter genes.

* PD-L1 nanobody

+ Normal cancer cells: have PD-L1 to “block” attacks from T-cells
+ PD-L1 nanobody: binds to PD-L1 so that cancer cells have no way to block attacks
+ Problem:

- requires immune cells to kill
- At later stages, immune system may be compromised
- Cannot kill cancer cells effectively

* Bax gene(2./2.1)

+ BCL2 associated X, apoptosis regulator
+ If Bax protein is transcribed, it promotes apoptosis (type of programmed cell death) in cancer cells

* Bax has been chosen

![](https://static.igem.wiki/teams/5441/notebook/img-2521-1.png)
[![](https://static.igem.wiki/teams/5441/notebook/img-2522-1.png)](#Apr Week 1)
[![](https://static.igem.wiki/teams/5441/notebook/img-2523-1.png)](#Apr Week 2)
[![](https://static.igem.wiki/teams/5441/notebook/img-2524-1.png)](#Apr Week 3)
[![](https://static.igem.wiki/teams/5441/notebook/img-2525-2.png)](#Apr Week 4)

![](https://static.igem.wiki/teams/5441/notebook/img-2584-1.png)

##### Week 1: Formulation of initial plan

1. Construction of plasmids
2. Purchase of A and B separately

1. A plasmid **containing our gene** that is **non-mammalian**; and
2. Another plasmid that is **mammalian** but **lacks any promoter genes** (pENTR1A)

* A and B will be digested, then ligated together using **BamHI** and **HindIII** enzymes
* Colony PCR → confirm size of plasmids are correct 1.e

1. Testing of PSMA promoter

* Evaluate the activity of PSMA promoter in different cell lines
* GFP (commonly used and known to work) as reporter gene

* ****Plasmid A: pENTR1A-PSMA-GFP****

* Transfected to cancer and non-cancerous cell lines

1. Testing of Gluc reporter gene

* Measure luminescence given out by reaction w/ Gluc as substrate

* ****Plasmid B: pENTR1A-PSMA-Gluc****

* Transfected to cancer and non-cancerous cell lines
* Testing of polymer delivery
* Make sure polymers can correctly transport plasmids to cancer cells

* ****Polymer plasmid conjugate X:****

* **PAMAM polymer**

* **A10-3.2 & DUP-1 aptamers**

* **Plasmid C: PB-Gluc**

1. Testing of killing function

* Confirm that Bax gene can kill cancer cells

* ****Polymer plasmid conjugate Y:****

* **PAMAM polymer**

* **A10-3.2 & DUP-1 aptamers**

* **Plasmid D: PB-Gluc-Bax**

![](https://static.igem.wiki/teams/5441/notebook/img-2585-1.png)

##### Week 2: Figuring out protocols used

1. Plasmid construction
   1. Plasmids in powder form: **transformed** into DH5a
   2. Colonies are picked → plasmids **extracted**
   3. **Restriction digestion:** linearise genes (BamHI and HindIII)
   4. **Gel electrophoresis**: visualise the results of digestion
   5. **Gel purification**: obtain the digested plasmids
   6. **Ligation:** stick two genes together
   7. **Colony PCR**: confirm that ligated genes have the correct size
2. Polymers
   1. PEG is attached to PAMAM to form **PAMAM-PEG** conjugate
   2. **Aptamers** are attached to PAMAM-PEG
   3. **BODIPY** are introduced to some of the PAMAM-PEG
      1. Microscopes are used to view BODIPY **fluorescence**
      2. Confirm that aptamers can bring plasmids to cells
   4. **Plasmids C & D** are introduced to other PAMAM-PEG
   5. **Polymer-plasmid conjugates** are mixed with cells
      1. MTT Assay are performed to quantify the death of cancer cells (required absorbance measurement)
      2. Luminescence of Gluc are detected

![](https://static.igem.wiki/teams/5441/notebook/img-2589-1.png)

##### Week 3: Required equipment and apparatus

* **Cell lines** ∵ environment for prostate cancers are hard to simulate with chemicals (purchased)
* **Plate readers:** measure luminescence (Gluc), fluorescence (GFP), absorbance (MTT assay) (equipped but only absorbance can be measured)
* **Live cells monitoring microscopes: monitor the situation of cell lines (equipped)**
* **PCR Thermal Cycler: perform colony PCR (equipped but is aged)**

![](https://static.igem.wiki/teams/5441/notebook/img-2587-1.png)

##### Week 4: Evaluation of initial plan

* ❓Construction of plasmids: colony PCR can be tried but cannot confirm that PCR machine will work
* Contingency plan: extract the ligated plasmids, digested using only 1 enzyme, then gel run to visualize the size
* ❓Testing GFP and Gluc light: required to borrow at other labs but possible
* ✔️Testing Bax killing function: feasible
* 🔜Polymers: the technical difficulty is too high

![](https://static.igem.wiki/teams/5441/notebook/sample-1.png)

![](https://static.igem.wiki/teams/5441/notebook/jul1.png)

![](https://static.igem.wiki/teams/5441/notebook/img-2579-1.png)

![](https://static.igem.wiki/teams/5441/notebook/aug1.png)

![](https://static.igem.wiki/teams/5441/notebook/aug2.png)

![](https://static.igem.wiki/teams/5441/notebook/sep1.png)

![](https://static.igem.wiki/teams/5441/notebook/sep2.png)

##### July-September Journal

Click [Here](https://static.igem.wiki/teams/5441/notebook/wet-lab-journal.pdf) to download PDF

## Game Development Notebook

##### March

* Defining gameplay style and features
* Programming the fundamental scripts
* Implementing some gameplay features

##### April

* Networking communications
* UI implementations
* Syncing player behavior between server side and client side
* Mobile support

##### May-June

* Game testing and balancing
* Bug fixing
* Server side http listeners

##### July-September

* Artwork implementation
* Code optimisation
* Adding additional features

##### Technical Issues

* Network module choices

+ Initially using netcode —> only support IP socket
+ Swap to mirror —> support Websocket

* Server setup

+ Initially using Docker —> complicated system for users without IT knowledge and difficult to replicate
+ Swap to using Node.js —> using only one file to handle http requests while only requiring to download Node.js module

* Syncing Server side and Client side

+ Using network identities in the mirror module
+ Reducing the use of rpc
+ Using formula to record FPS and Ping

* Device identification

+ WebGL doesn’t support device identification
+ Using manual UI input for players to choose

* Browser support

+ Using Unity WebGL

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## Engineering cycle

We always follow the design-build-test-learn cycles to move the project forward. This year’s project is
full of turbulence but we finally completed our project with a satisfactory result. The project can be roughly
divided into several cycles, which will be mentioned in the following paragraphs.

## Cycle 1: From protein level to genetic level

##### Design

Based on the research papers by Vlachostergios et al. (2021), we **chose prostate-specific
membrane antigen, PSMA**, as the biomarker for our project. PSMA is a protein found on the
surface of prostate cancer cells discovered in recent years. It is one of the newest and most accurate
biomarkers used in the detection and treatment of prostate cancer. (For more, please refer to the [history of prostate cancer](description) on the description page). Given the fact that there is
a high frequency of PSMA expression in all stages of prostate cancer (Minner et al., 2010; Tsourlakis et al.,
2015), it has a higher correlation for prostate cancer than PSA, another biomarker commonly used.

Based on the experience shared by the iGEM team last year, (referring to our [integrated human practice](human-practices)) protein purification typically results in a lower
antibody yield, which makes it hard to verify the result. Apart from that, the concentration of PSMA in
patients’ urine was unknown, making it tough to know the threshold value for detection. Thus, we decided
against constructing a protein. Instead, we utilised **another pathway with a similar concept to
detect prostate cancer** this year.

The pathway we used to **detect prostate cancer uses plasmids, containing PSMA
promoters**, which will be transported to the cells in the prostate gland. To produce PSMA,
prostate cancer (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
promoters in our plasmid and transcribes our required genes to detect and kill cancer cells.**

At first, green fluorescent protein (GFP), a protein that exhibits green fluorescence when exposed to
light in the blue to ultraviolet range, stood out to our eyes. It was a commonly used gene with easy-to-record
results, which can be **easily excited using blue light and observed** under an
inverted microscope. Utilising these characteristics, we can use GFP to **evaluate the
proficiency of the promoter** of our chosen biomarker, **PSMA**.

For more information regarding the design of the plasmid, please refer to our [Description](description) page.

Aim: to build a plasmid (pENTR1A-PSMA-GFP) that allows us to detect PSMA-positive prostate cancer cells
by measuring the fluorescence by GFP.

##### Build

We purchased the pENTR1A backbone and another plasmid containing the PSMA promoter and GFP gene. Both
plasmids were digested and gel electrophoresis was performed.

We then purified the agarose gel and the two genes were ligated to form plasmid pENTR1A-PSMA-GFP. (for
more information refer to [cycle 3](#cycle3))

The ligated plasmids were then transformed into competent DH5a and extracted. They were digested to
confirm that the gene length was correct.

After the ligated plasmids were extracted, we transfected different concentrations of them into multiple
concentrations of PSMA-positive cancer cell line MLLB-2.

##### Test

Fluorescence given out by GFP was visualised by using an inverted microscope with a live cell-monitoring
system.

###### First attempt

We collected the cell media of the cancer cells containing transfected plasmids, which were then
photographed with an inverted microscope. At first, we recorded a bright image with a lot of light-emitting
spots. However, it was suspected to be a false positive result, and the reasons are evaluated below.
themselves.

###### First attempt evaluation

We have discussed the results with Dr Yu, who has extensive experience in GFP analysis. (For more, please
refer to our [integrated human practice](human-practices) page)

Possible reasons for the false positive result:

1. Most of the spots are a result of **autofluorescence** from the cell
   organelles but not GFP.
2. Only the cell media was photographed, which mostly consisted of **dead cells that could
   not transcribe genes.**

Possible reasons why fluorescence from GFP is not observed:

1. There was a noticeable delay between collecting the supernatant and photographing, which may lead to
   the **degradation of GFP.**
2. The **concentrations** of plasmids/cells may be **too
   low**, which resulted in not enough GFP being expressed and seen.

![](https://static.igem.wiki/teams/5441/cycle/c1f1.jpg)

Cell media from cells with pENTR1A-PSMA-GFP photographed with a blue light filter

###### Second attempt

After evaluating the previous attempt, we made the following changes:

1. Photograph cells directly instead of cell media
2. Cells were kept in a 37°C incubator with 5% CO2 until minutes before photographing to prevent the
   degradation of GFP.
3. Cells were photographed in the same position with and without the blue light filter to confirm the
   fluorescence given out by actual cells.

We have successfully photographed the fluorescence given out by GFP genes transcribed by
pENTR1A-PSMA-GFP.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5441/cycle/c1f2.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c1f3.jpg) |
| Cancer cells photographed in a live cell monitoring system | The same cells photographed with a blue light filter |

##### Learn

From the process of using a **fluorescence microscope** to visualise the
results of transcribed GFP, we learnt that

1. Cells should be photographed directly instead of using cell media
2. Photographs must be done **rapidly** after the cells are taken out of the
   incubator

Moreover, we confirmed the success of converting from a protein level to a genetic level. Unlike protein
synthesis, plasmid
constructions **are easily replicable**, and we were able to **not only
discover issues earlier but also synthesise a high concentration of plasmids**. By
purchasing:

1. A plasmid that contains our gene but is not mammalian; and
2. Another plasmid that is mammalian but does not contain any promoter genes,

We constructed our plasmid by combining A and B mentioned above. **Purchasing prefabricated
plasmids (A+B) from firms like Genscript is ideal for long-term storage** as it comes in powder
form. However, this method is **immensely expensive**. Our approach of purchasing
**A and B separately is less costly**, which is **more practical for our
project**. This approach also allows for more flexibility in plasmid construction as we could mix
and match these genes for use in other formulations, i.e. combining B with another gene that we would use.

## Cycle 2: Changing reporter gene

##### Design

We **initially chose GFP as the reporter gene** for our project. However, it
has quite a **few disadvantages**. For one, it is
**cytotoxic** (Ansari et al., 2016) and requires external excitation light, which is
**harmful** and **not possible when it comes to** ***in
vivo*** **studies**. For another, autofluorescence from cells and tissues can
interfere with the GFP signal, making it **difficult to distinguish true GFP
signal** from background noise.

Knowing the disadvantages of GFP, we realised that another reporter gene must be used for future
development. Hence, after countless literature readings, we **selected Gaussia luciferase (Gluc)
as our reporter gene**. Gluc is a 20kDa protein from the marine copepod, Gaussia princeps,
**which is a bioluminescent enzyme that is highly secreted into urine and can be detected through
it** (Tannous, 2009). Gluc provides the following advantages:

1. It produces a **strong bioluminescent signal**, making it extremely
   sensitive for detecting low levels of activity.
2. Does **not require external excitation light**, making it
   **suitable for** ***in vivo*** **studies**.
3. Gluc is naturally secreted into the media when performing cell culture, which makes it
   **less arduous**.

As Gluc is secreted into the urine, in the future, we hope to be able to collect urine samples of
patients, which will allow for longitudinal studies and repeated measurements over time on the level of cancer
cells in a patient.

##### Build

###### Plasmid construction

We purchased the pENTR1A backbone along with another plasmid that contains a PSMA-Gluc gene.

After digesting both plasmids, we performed gel electrophoresis. We then purified the agarose gel and
ligated the two genes to create the plasmid pENTR1A-PSMA-Gluc (for more details, see cycle 3). The ligated
plasmids were transformed into competent DH5α cells and subsequently extracted. To confirm the correct
gene length, we digested the extracted plasmids.

###### Plasmid construction

Following this, we transfected various concentrations of the
plasmids into different amounts of the PSMA-positive cancer cell line MLLB-2, which were subcultured into
96-well plates one night beforehand. The cell lines are incubated for 2 more days in a 37°C environment
with 5% CO2.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5441/cycle/c2f1.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c2f2.jpg) |
| Plasmid map of pENTR1A-PSMA-GFP | Plasmid map of pENTR1A-PSMA-Gluc |

##### Test

Following that, Gaussia Luciferase Flash Assay was performed to start the oxidation of coelenterazine
with Gluc as a substrate. Cell media is collected to measure the secreted Gluc specifically. The luminescence
in the cells was quantified with a plate reader.

###### Attempt 1

We set the plate reader to measure the luminescence given out at a specific wavelength 485nm. (Emission
maximum of the assay)

![](https://static.igem.wiki/teams/5441/cycle/c2f3.jpg)

Luminescence given out by different combinations of plasmid and cell concentrations. (P3>P2>P1,
C3>C2>C1)

###### Attempt 1: Evaluation

Our team had the opportunity to interview Dr. Molly Wenqian Xue, a Technical Specialist and Assistant
Manager at ThermoFisher Scientific. We learnt that as we are using a flash assay, luminescence at
**all wavelengths** should be measured instead of 1.

###### Attempt 2

We fixed the problem from the previous attempt and measured luminescence at another setting.

![](https://static.igem.wiki/teams/5441/cycle/c2f4.jpg)

Settings for the measurement from attempt 2. Blanks are set to wells with only cell media without any
plasmids.

###### Attempt 2: Evaluation

![](https://static.igem.wiki/teams/5441/parts/gluc-results-line.jpg)

100,000 cells/well

At 100k cells per well, change in plasmid concentration did not show significant differences for
luminescence measured. Moreover, the luminescence measured is lower than the blank samples, which shows signs
of contamination.

Evaluation (100,000 cells/well)

The cell concentration may be too high, leading to nutrient competition in the medium of cells. As a
result, the final number of cells may not be accurate and thus the correlation between plasmid concentration
and luminescence cannot be correctly shown.

10,000 - 50,000 cells/well

At lower concentrations of cells per well, no matter the plasmid concentration, the luminescence measured
is always **higher than the blanks.**

Evaluation (100,000 cells/well)

This shows a positive result of detection for PCa using our method, for all concentrations of PCa will be
able to be detected with the minimum concentration of plasmids used in the test.

##### Learn

96-wells have a limiting capacity for growing cells, implying that more cells do not necessarily relate
to higher levels of luminescence. The cells may compete for nutrients and die as a consequence, leading to
inaccurate numbers of cells, thus the correlation between the number of cells and plasmid concentrations could
not be visualised. Therefore, we realised that we should use lower concentrations of cancer cells in future
experiments.

Moreover, our minimum concentration of plasmids was able to detect the lowest PCa concentration used
(10,000 cells/well), demonstrating a high sensitivity for our product, which could allow early stages of
prostate cancer to be detected and cured promptly.

## Cycle 3: Killing cancer cells

##### Design

After the success of cycles 1 and 2, we moved on to the core part of our project: killing prostate cancer
cells promptly after detection. At first, referencing results from other teams
(*Tsinghua - IGEM 2023*, n.d.), we planned to use
PD-L1 nanobody, an antibody to target immune checkpoints, enhancing T-cell response and mediating antitumor
activity by cancer cells. However, this method relies on the immune system, which is an additional unknown
factor for patients.

Therefore, we finally settled on the apoptosis regulator BAX, also known as bcl-2-like protein 4. The
BCL-2 family of proteins, including pro-apoptotic proteins like BAX, play a crucial role in regulating
apoptosis (programmed cell death) (Cory et al., 2016). They control mitochondrial
dysfunction and cell killing, and we utilised this characteristic of BAX to kill cancer cells in our
project.

As concluded from the results of cycles 1 and 2, low to medium concentrations of cells are the easiest to
detect. Therefore, we transfected different concentrations of plasmid into 20,000 cancer cells for each
well.

##### Build

The transfected cancer cells were continuously incubated in a 37°C incubator with 5%
CO2 throughout the experiment in RPMI 1640 medium. We monitored the cell viability
over time, including a control without any cells transfected, using a CyQUANT™ MTT Cell Viability Assay.
It employs the widely-recognized MTT reagent to assess mammalian cell viability. In active mammalian cells,
the redox potential reduces MTT to a vividly coloured formazan product, which can be measured using a
microplate absorbance reader.

##### Test

![](https://static.igem.wiki/teams/5441/result/bax3.png)

Cell viability across three days after pENTR1A-PB-Gluc-Bax transfection into MLLB-2 cells

During the three days of measurement, we successfully recorded a decrease in cancer cell proliferation
rate, compared to the control which has no plasmid transfected.

##### Learn

Expressing Bax is a successful method for inhibiting growth of prostate cancer cells and even reducing
their number. Refer to our **Results** page for more information regarding the
result.

## Cycle 4: Plasmid construction (Web-Lab) on Practical Basis

##### Design

We followed a set of standard protocols, found in manufacturers’ product sheets, during our process
of plasmid construction.

##### Build-1

During the first step of plasmid extraction, we **incubated the colonies for 8
hours** and **centrifuged the bacterial cells for only 1 time**.
However, the **pellet was not obvious with only a tiny size**. The extracted
plasmids had a **deficient concentration**, and further operations such as digestion
failed with poor banding observed.

![](https://static.igem.wiki/teams/5441/cycle/c4f1.jpg)

Poor result in gel electrophoresis

##### Test-1

After consulting other more experienced teachers in the field, we tried to **centrifuge the
colonies multiple times**, by discarding the supernatant after each centrifuge step and then
**adding more bacterial culture**. This resulted in a noticeable increase in the size
of the pellets, and we used a Multiskan SkyHigh Microplate Spectrophotometer to measure the concentration of
plasmids afterwards. We successfully achieved an **incredibly concentrated volume of
plasmids**, which made downstream operations much easier.

![](https://static.igem.wiki/teams/5441/cycle/c4f2.jpg)

Increase in size of pellet

![](https://static.igem.wiki/teams/5441/cycle/c4f3.jpg)

Figure 5: Result of measuring the concentration of plasmid

##### Build-2

For another, when we first performed restriction digestion, no measurement of the concentration of
extracted plasmids was taken. We only **followed the suggested volumes given by the
manufacturer**, resulting in a **very dim band** after gel
electrophoresis. After gel purification, only a very small amount of DNA remained (measured with a
spectrophotometer). **Ligation of the said DNA resulted in no visible colonies after
transformation**.

![](https://static.igem.wiki/teams/5441/cycle/c4f4.jpg)

Dim banding in gel electrophoresis after restriction digestion

##### Test-2

With the **measurement from the spectrophotometer,** we were able to
**calculate the volume of DNA required for a specific mass**, leading to us being able
to add a **suitable amount of DNA** for enzymes to digest. Also, we
**scaled up the reaction to prevent possible pipetting errors**. After making the
adjustments, we were able to **obtain bright bandings in gel electrophoresis** which
were suitable for gel purification. However, as the difference of base pairs of the plasmid before and after
cutting was a mere 100bp, we failed to separate digested and undigested plasmids and had to cut the entire
banding.

![](https://static.igem.wiki/teams/5441/cycle/c4f5.jpg)

Bright banding in gel electrophoresis after restriction digestion

##### Build-3

After purifying the DNA and then ligating it, we transformed the resulting plasmids into bacteria.
Colonies were able to grow, which indicated that either a) **our plasmid has been successfully
ligated** or b) undigested pENTR1A were transformed.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| ![](https://static.igem.wiki/teams/5441/cycle/c4f6.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f7.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f8.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f9.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f10.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f15.jpg) |
| pENTR1A-PSMA-Gluc | pENTR1A-PSMA-Gluc | pENTR1A-PSMA-GFP | pENTR1A-PSMA-GFP | +ve control (plasmid) | -ve control |

Bacterial transformation of different plasmid

We performed colony PCR screening to ensure the GOI had been successfully implanted into the
plasmid.

##### Test-3

However, when we performed gel electrophoresis, we found out that the method **could not
successfully show if we had successfully combined our GOI with the plasmid**. We could only see a
very dim band that remained stuck in the well after neglecting the bands formed by the primers.

![](https://static.igem.wiki/teams/5441/cycle/c4f11.jpg)

Result of colony PCR, visualised by performing gel electrophoresis

We believe that one of the following reasons occurred: **(i) incorrect extension time; (ii)
incorrect primer; and (iii) faults in the PCR machine**. However, we cannot figure out which of
them is the real reason.

##### Build-4

We have **chosen to use another method to verify our results in plasmid
construction**, which is to extract the plasmids and then digest them to visualise their
size.

##### Test-4

Gel electrophoresis were run to visualise the **size of plasmids**. Then we use
a restriction enzyme to cut the plasmid to check whether the desired plasmid is being cloned successfully. All
of them had the correct size, which means that the **colonies contained successfully ligated
plasmids**.

|  |  |  |
| --- | --- | --- |
| ![](https://static.igem.wiki/teams/5441/cycle/c4f12.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f13.jpg) | ![](https://static.igem.wiki/teams/5441/cycle/c4f14.jpg) |
| Undigested plasmids (supercoiled)  Proved that at least some plasmid being ligated successfully | Digested (Using 1 restriction enzyme to cut the plasmid to check the base pair) pENTR1A-PSMA-GFP and pENTR1A-PSMA-Gluc | Digested pENTR1A-PB-Gluc-Bax |

Digestion results of ligated plasmids

##### Learn

From this cycle, we have learnt that the protocols from the manufacturer may not fit our project. We may
need to modify them to obtain success in constructing our plasmid, including changes in:

1. **Size of bacterial pellet** during plasmid extraction
2. Add DNA for digestion in terms of **mass** not volume

We also have to change our methods for verifying the ligated genes because of faults. For example, colony
PCR was changed to a more traditional way of restriction digestion since we cannot confirm the reason of it
failing.

## Cycle 5: Cell culture

This year, we have chosen two cell lines, **MLLB-2** and
**PNEC30**, to verify the result of our project. Hence, this cycle focuses on how we
handle the cells before ascertaining the result of plasmid construction.

##### Design

Due to the decision to apply plasmid to cells, we initially decided to purchase **several
human cell lines** to investigate the **fate** of the plasmid and the
**response** of cells. However, due to safety concerns and regulation issues, we have
finally chosen **two mammalian cell lines, originating from rats,** to be our
crucial components of result verification.

We have found a PSMA-**positive** mammalian cell line, MLLB-2, from the website
of the American Type Culture Collection (ATCC). However, we could not find a PSMA-negative cell line at the
beginning. After searching for more information, we finally figured out that neuroendocrine prostate cancer
cells do not have PSMA expression (Bakht et al., 2019). Therefore, we have chosen PNEC30, which
is a **neuroendocrine prostate cancer cell line**, as the other cell line. We have
decided to follow **ATCC’s protocol** to handle our cells.

##### Build-1

After the arrival of cell lines, we thawed and cultured them in several T-flasks. MLLB-2 was cultured in
RPMI 1640 medium while PNEC30 was cultured in neural progenitor maintenance medium (by Lonza)

##### Test-1

After a day, we observed the two cell lines under an inverted microscope. While MLLB-2
**grew well** in the T-flask, PNEC30 grew with a much **lower cell
confluency** in the flask. We even saw a **blurry object floating on the
surface** of the PNEC30 medium. Therefore, after discussing with our instructor, Ms Ng, we have
decided to aspirate the object away to another flask and grow it in another incubator with the same settings
to investigate what the object is.

A day after removing the blurry object from the flask, PNEC30 **grew normally**while the blurry object grew much larger, and some of the objects stuck to the bottom of the
flask, forming a thick layer. We suspected that the presence of blurry objects was due to contamination, and
we believed that it was due to fungal infection after seeking professional advice.

![](https://static.igem.wiki/teams/5441/cycle/c5f1.jpg)

MLLB-2 a day after thawing

![](https://static.igem.wiki/teams/5441/cycle/c5f2.jpg)

A blurry object floating on the surface of the PNEC30 medium.

![](https://static.igem.wiki/teams/5441/cycle/c5f3.jpg)

Suspected fungal contamination in PNEC30

![](https://static.igem.wiki/teams/5441/cycle/c5f4.jpg)

The blurry object in an isolated flask after a day

![](https://static.igem.wiki/teams/5441/cycle/c5f5.jpg)

PNEC30 after separation of the blurry object

##### Learn-1

As we found that the presence of blurry objects is due to **fungal infection**,
we have paid more attention to our aseptic techniques since then. We then used 70% alcohol more frequently to
**sterilise every component** that we put into the biosafety cabinet **in
detail** and have also **sterilised our arms more thoroughly** before
operating in the biosafety cabinet after this accident.

##### Build-2

We have then handled the cell lines with care. We changed the medium **every two days**and subcultured the cells to another flask when the cell confluency was above 85%.

![](https://static.igem.wiki/teams/5441/cycle/c5f6.jpg)

PNEC30 with a cell confluence of 85% after recovery from contamination

##### Test-2

MLLB-2 was ready for subculturing. However, we decided to wait for a day more. On the next day, the
confluency of MLLB-2 decreased and they started to detach from the bottom and clump up, we instantly carried
out the first subculture of MLLB-2 and also cryopreserved a portion of it. After a few days, we observed the
subcultured cells with an inverted microscope and discovered that the cell line grew rapidly in number but
**did not stick well** to the bottom of the flask and had **many cell
clumps**.

![](https://static.igem.wiki/teams/5441/cycle/c5f7.jpg)

MLLB-2 before subculture (detached and clumped up)

![](https://static.igem.wiki/teams/5441/cycle/c5f8.jpg)

MLLB-2 few days after subculturing

##### Learn-2

With the **decreased cell confluency** before
**subculturing**, we learnt that we should subculture cells instantly when the
confluency reaches 85%. As for the cell clumping and poor attachment issue, We rethought our procedures and
concluded that it was because we **did not pipette up and down thoroughly enough to respond to
the cells after centrifugation**. Through this incident, we have learnt that we need to pipette
up and down more thoroughly in the next subculture.

##### Build-3

After **seeding MLLB-2 cells** into 96-well plates for the experiment, we
transfected plasmids pENTR1A-PSMA-Gluc into the cells, experimenting with the effect of different plasmid
concentrations on different concentrations of cells.

##### Test-3

After measuring the **luminescence** of the reaction between
**coelenterazine**(as substrate) and **Gaussia
luciferase**(as an enzyme), the result shows that the plasmid was transfected in excess, causing
the cell concentration to become the **limiting factor of the experiment**. However,
we are investigating the **optimal concentration** of dna for the most efficient and
**accurate detection** of prostate cancer cells, so concentration of dna should be set
as the **limiting factor** in the experiment.

##### Learn-3

With the result, we learnt that we should reduce the concentration of Gluc dna in transfection, this
causes the cell concentration to become the limiting factor. Therefore, in the next transfection of Gluc, we
decided to reduce the amount of dna added while **diluting it to the same total
volume** as before. A correct graph was obtained.

##### Build-4

At the first reading of the luminescence of Gluc, we used plate reader to measure our data.

##### Test-4

In the reading process, we used a wrong setting for the plate reader. Therefore, the result obtained was
not in the optimal format and was inaccurate

##### Learn-4

After searching for the manual of the plate reader, we found the **correct mode for the
luminescence detection of Gluc**. We then repeated the transfection of Gluc into
**newly seeded MLLB-2 cells**, and read the luminescence of Gluc again, correct data
were obtained.

{% endblock %}

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{% block title %}Experiments{% endblock %}
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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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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**](https://parts.igem.org/Part%3ABBa_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**](https://2024.igem.wiki/plklfc/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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-1.jpg) |
| **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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-2.jpg) |
| **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.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-3.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-4.jpg) |
| **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 |

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-5.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-6.jpg) |
| **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 |

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-7.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-8.jpg) |
| **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 :

|  |  |  |
| --- | --- | --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-9.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-10.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-11.jpg) |
| **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**](https://parts.igem.org/Part%3ABBa_K5441012)). Our experimental design can be reviewed at [**Design**](https://2024.igem.wiki/plklfc/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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-12.jpg) |
| **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.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-13.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-14.jpg) |
| **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 mm****²) and low-concentration (10,000 cells** **per 0.33 mm****²) 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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-15.jpg) |
| **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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-16.jpg) |
| **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 mm****²)**, 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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-17.jpg) |
| **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.

|  |
| --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-18.jpg) |
| **Fig 13.** Broken line chart of absorbance by MTT cell assay under different plasmid concentrations at different days |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-19.jpg) |
| **Fig 14.** Bar chart of absorbance by MTT cell assay under different plasmid concentrations |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-20.jpg) |
| **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.

|  |  |  |  |
| --- | --- | --- | --- |
| ![](https://static.igem.wiki/teams/5441/resultpics/result-21.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-22.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-23.jpg) | ![](https://static.igem.wiki/teams/5441/resultpics/result-24.jpg) |
| 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 | | | |

{% endblock %}