iGEM 2023 — Team patras

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#### Week 1 (July 12 - July 18, 2023): Lab Training - Basic Techniques

* **Day 1 (July 12)**: Basic Laboratory Safety Training:
  Safety protocols, lab equipment usage, and emergency procedures.
* **Day 2 (July 13):**: Cell Culture Training:
  Introduction to aseptic techniques, cell culture setup, and maintenance.
* **Day 3 (July 14)**:Protein Extraction Training:
  Techniques for protein extraction and quantification.
* **Day 4 (July 15)**:SDS-PAGE and Western Blotting Training:
  Gel electrophoresis, Western blotting setup, and antibody incubation
* **Day 5 (July 16)**:Data Analysis Training:
  Introduction to data interpretation and analysis software.
* **Day 6-7 (July 17-18)**:No lab procedures were performed

#### Week 2 (July 19 - July 25, 2023): Lab Training - Advanced Techniques

* **Day 8 (July 19)**:Advanced Cell Culture Techniques:
  Subculturing, transfections, and specialized culture conditions.
* **Day 9 (July 20)**:Molecular Biology Techniques:
  DNA/RNA extraction, PCR, and gene expression analysis.
* **Day 10 (July 21)**:Advanced Western Blotting:
  Troubleshooting, quantification, and multiple antibody techniques.
* **Day 11 (July 22)**:Microscopy and Imaging:
  Troubleshooting, quantification, and multiple antibody techniques.
* **Day 12 (July 23)**:Biosafety and Biosecurity:
  Advanced safety procedures and regulations.
* **Day 13-14 (July 24-5)**:No lab procedures were performed

#### Week 3 (July 26 - August 1 , 2023): Initial Cell Cultures

* **Day 15 (July 26)**:Cell Culture and Establishment:
  + Prepare reagents and materials.
  + Seed cells into culture flasks/plates.
* **Day 16 (July 27)**:Initial Culture Maintenance:
  + Check cell culture.
  + Exchange half of the medium.
* **Day 17 (July 28)**:Ongoing Culture Maintenance.
  - Continue culture maintenance.
* **Day 18 (July 29)**:Cell Harvest and Freezing:
  + Detach cells from plates.
  + Pellet cells and freeze.
* **Day 19 (July 30)**:Cell Harvest for Protein Isolation:
  + Harvest cells for protein isolation.
  + Quantify protein concentration.
* **Day 20 (July 31)**: Western Blotting - Block and Antibody Incubation (Day 1):
  + Prepare samples.
  + Run SDS-PAGE gel.
  + Transfer proteins to a membrane.
  + Block the membrane.
* **Day 21 (August 1)**:Western Blotting - Washing and Antibody Incubation (Day 2):
  + Wash the membrane.
  + Incubate with primary and secondary antibodies.
  + Develop the Western blot.

#### Week 4 (August 2 - August 8, 2023): Cell Establishment and Early Monitoring

* **Day 22 (August 2)**:Cell Establishment - Acquisition and Isolation:
  + Collect samples.
  + Isolate cells.
* **Day 23 (August 3)**:Cell Establishment - Isolation of Cells:
  Isolate mononuclear cells.
* **Day 24 (August 4)**:Cell Establishment - Isolation of Cells (contd.):
  Wash and count the isolated cells.
* **Day 25 (August 5)**:Cell Establishment - Resuspension:
  Resuspend cells in culture medium with 20% FBS.
* **Day 26 (August 6)**:Cell Establishment - Viability Assessment:
  Count cells and determine their viability.
* **Day 27 (August 7)**:Cell Establishment - Storage:
  + Prepare cells for storage with DMSO.
  + Freeze and store cells for future use.
* **Day 28 (August 8)**:Cell Proliferation and Monitoring:
  + Continue to maintain cell culture.
  + Exchange medium as needed.
  + Observe cell proliferation.
  + Cells were frozen on 80°C

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**Weeks 5-12**:Experimental Continuation and Article Review

* Conduct additional literature review for updated insights.
* Incorporate new findings into the project.

#### Week 13: (October 4-October 10, 2023) Cells get unfrozen and cultivated,Western Blotting

* **Day 85 (October 4)**:Cell Plating and Initial Culture:
  + Plate cells on cell culture plates.
  + Incubate at 37°C with 5% CO₂ for 48 hours (2 days).
* **Day 86 (October 5)**:Experimental Continuation:
  Continue monitoring cell growth and experiments.
* **Day 87 (October 6)**:Washing and Differential Medium:
  + Wash cells with PBS.
  + Divide cells into different media as per protocol.
* **Day 88 (October 7)**:Experimental Continuation:
  Continue experiments and data collection.
* **Day 89 (October 8)**:Cell Harvest and Freezing:
  + Harvest cells as per protocol.
  + Pellet cells and freeze for future use.
* **Day 90 (October 9)**:Experimental Continuation:
  + Resume experiments with unfrozen cells.
  + Continue data collection.
* **Day 91 (October 10)**:Western Blotting:
  + Block and Antibody Incubation (Day 1 of Western blotting) as per the provided protocol.
  + Begin the Western blotting process.
* **Day 92 (October 11)**:Data Analysis and Recording:
  + Secondary Antibody Incubation (Day 2 of Western blotting)
  + Western Blotting Development
  + Analyze the data collected from the experiments.
  + Record and document the results for future reference.

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#### Design

Our plan was to create a biosensor that could be used as an affordable yet accurate and reliable diagnostic tool for oncologic applications using bioengineering methods. After carefully reviewing the latest scientific literature on biomarkers, we concluded that, given the evidence, the best option would be the assessment of the Phosphorylation Level of Mitogen Activated Protein Kinases (MAP Kinases).

As their name implies, kinases, phosphorylate specific groups, and by an extend control the activity and function, of biomolecules consisting a cascade crucial for cellular processes such as proliferation, differentiation, death and overall survival, utilizing a phosphate group originating from Adenosine Triphosphate (ATP). Under normal conditions, MAPs are activated by other kinases in response to a cellular signal by alteration of the phosphorylation level, however in phenomena that lead to oncogenesis and eventually cancer, due to alterations in the physiology of the cell, their phosphorylation state is deregulated. Thereby, by measuring said state in a tissue, it is plausible to assess the possibility of cancer occurrence.

We considered kinase phosphorylation as a valuable biomarker for cancer as they provide significant evidence for the alteration of cell physiology and oncogenesis. There are many existing proteomic methods to assess such metrics such as western blot and mass spectrometry, however each one of them had a combination of shortcomings that abstract their commercial application and scaling including amongst others high cost of operation and reagents, duration and personnel training. Therefore we began considering and looking for alternatives that would address said issues in order to streamline early, affordable and precise cancer diagnosis. Having searched through the scientific literature, we found out that such device would probably come in form of a biosensor like setup, able to recognise the phosphorylated kinases.

In order for the assessment to be successful we needed to find the right method to detect and measure the phosphorylation state, or in other words kenome. Detection of kinases molecules for the profiling of the sample can be done using engineered antibodies that bind to them. For the sake of making the method time efficient and economical, we have opted to proceed with micro-wells to study the reaction that leads to the detection. On the walls of each micro-well of an array, affimers, a more efficient alternative to antibodies that are smaller, have a wider range of target molecules and a faster production rate that does not require animals, are placed and bind to target kinases. After washing the unbound molecules, enzymes remove phosphate and reactants that illuminate once in contact with it thus granting the ability for quantification.

#### Build

Once we had accumulated the necessary information, we proceeded with applying our research to a device that would eventually be the diagnostic tool. Our build was based on an existing patent designed for the detection of SARS-CoV-2 using ACE-2 receptors co-developed by our Principal Investigator. The biosensor is primarily consisted of two components, one physical/biological were the reactions take place and one electronic were the detection and user interface happens and the reaction as well as all the parameters are controlled. Those two components are connected to each other via gold electrodes to minimize resistance to the current and all the implications that it would result such as degradation of biomolecules due to temperature and inaccurate results. The electrodes’ surface needed to be clean to minimize contamination and maximize durability and accountability. After cleaning, L-Cysteine were applied to the electrodes for the bonding of massive biomolecules to the gold electrodes via their thiol groups. Binding to the L-Cysteine layer are the affimeres used for the detection of kinases, after proper preparation. Those affimeres are specific to the kinases that we want to detect and alter the physicochemical characteristics of the solution when bound to them. The biosensor utilizes the alteration of the electrochemical properties of the solution to sense the presence as well as the state of the reaction. We also used Wi-Fi and Bluetooth connectivity in order to implement Internet of Things protocols thus making the process more efficient and scalable.

![brochure](https://static.igem.wiki/teams/4639/wiki/gr1-lrg.jpg)

#### Test

To test the biosensor, we will collect and test blood samples from a population of 30 patients and 5 healthy donors under the guidelines set by our bioethics committee approved trial protocol, since human biological material raises special bioethical and legal concerns. Although the biosensor can, under conditions, be used for specimens originating from any tissue, for our experiments we have optimized the device for blood as it is the most easily extractable and manageable tissue whilst maintaining diagnostic value. Sampling will be carried out by medically trained personnel at the controlled and hygienic facilities of “Errikos Dynan” hospital in Athens, Greece and handling will be a responsibility of trained members of our team originating from the health and life sciences sector. Our protocol binds the team to treat the donors as well as handle the samples and the generated data with the highest respect. Additionally, we grant the participants the ability to recall their participation as well as the right to be forgotten. Should any breach to the guideline of the protocol occur, testing will pause and the case will be examined and if needed the project will be terminated prematurely.

#### Learn

As was expected, the team had faced some setbacks in several aspects of the project. One of our most significant challenges was fundraising for the project as well as sourcing the affimers and other reactants for the build. We believe that such a situation was faced due to the saturation of the sector leading to greater competition for supplies as well as the dire financial state many institutions and other parties are facing.

Additionally, we allocated significant resources and consideration towards the handling of the biological material as we intended to follow Good Laboratory Practice in accordance with bioethical and biosafety standards in order to protect the identity of the donors, the health and safety of our members and the wider community and the integrity of our experiments.

In the event that our observations do not match with the expectations originating from our theoretical background, we will have to retrospect on all our processes and likely points of error. We will also review our theoretical background and plan of action in order to assess any possible shortcomings or oversights.

![brochure](https://static.igem.wiki/teams/4639/wiki/engineering-cycle-infinite-removebg-preview.png)

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#### Sensor Coating with ORDYL SY 300

Gold interdigitated electrodes (IDEs) were purchased from DropSens (Asturias, Spain), (cat. N.: PW-IDEAU50). Each IDE has a finger width and spacing of 50 μm, with a total number of 70 fingers, a total electrode length of 7 mm, and electrode surface area of 8.45 mm2. Initially, the IDEs were immersed in an acetone solution, then in 100% isopropanol and finally, they were washed with distilled water. (Chemicals were purchased from Sigma-Aldrich (St. Louis, CA, USA).)

For sensor coating, it was chosen to use dry resin, specifically ORDYL SY 300, which is a permanent dry film with a very high possible aspect ratio and excellent high thermal and chemical stability. The experimental procedure is shown in the following Figure; The IDEs were placed in a hot plate at 110 °C. An ORDYL SY 300 piece, slightly larger than the IDE sensitive area, was cut, ORDYL protective liner was removed from the one side and the tape was coated at the IDE surface. A photolithography mask was designed and printed. The sample was exposed at UV for 48 sec (0.8 min The white dark areas of the mask protected the photosensitive layer from UV, whereas the dark transparent areas were exposed. The ORDYL protective liner was removed from the other side, revealing its photosensitive surface. Then, it was immersed in its developer solution, in an ultrasonic bath, for another 5 min. As a result, the ORDYL SY 300, being negative photoresist, was removed from the areas protected from the UV exposure. After the developing process, the sample was immersed again in 100% isopropanol solution and washed with distilled water. At the end, it was placed at a hot plate at 120 °C for 45 min, in order to harden the ORDYL layer. The thickness of the ORDYL layer before the post baking process was 90 μm.

![brochure](https://static.igem.wiki/teams/4639/wiki/experiments.png)

#### Grafting Antibody on Interdigitated Gold Surface

To clean the electrodes' surface, interdigitated capacitors prepared with ORDYL SY 300 were immersed in a solution of 100% isopropanol for 5 min and then rinsed in Milli-Q water. 30 μL (25 mM) of L-Cysteine were placed on the electrode surface and let to form bonds between gold and the amino acid’s thiol groups. The reaction was stopped when all L-cysteine solution dried out and three washes were performed using PBS to remove unbound material. Subsequently, a solution containing 5 μL (50 ng/ml) of ERK antibody, 5 μL (0.877 g/ml) of EDC and 10 μL (0.1 M) of MES was placed on top of the L-Cysteine layer. Specifically, EDC and MES activate the carboxylic terminus (-COOH) of ERK antibody. Once active the carboxylic terminus of ERK antibody reacts with the amine group (-NH2) of L-Cysteine forming a covalent peptide bond. Due to the cross reactivity between C- and N- termini of both ERK antibody and L-Cysteine, unwanted covalent bonds were formed also among the C-terminal of L-Cysteine and the N-terminal of ERK antibody. After each step, the biosensor was rinsed 3 times in Milli-Q water, in order to remove unbound molecules from its surface. The IDE with immobilized ERK antibody or L-Cysteine alone was covered with 20 μl of PBS and kept wet until its use. Some of the biosensors were kept standby for 7 days at 4 ◦C in order to be used for the biosensor stability calibration.

#### Cell Culture

The Α453 cell line was maintained in DMEM medium, containing 10% fetal bovine serum (FBS), 1X penicillin, 1X streptomycin, and 2 mM L-glutamine. Cells were incubated at 37°C, 5% CO2 in a humidified atmosphere. A453 cell lines used in the study have been purchased from American Type Culture Collection (ATCC).

#### Western Blot and Antibodies

Total protein was extracted with 60 mL RIPA lysis buffer and Western blotting was performed according to standard protocols. Blots were incubated overnight at 4°C with the appropriate primary antibodies. Protein extracts have been prepared from at least two independent experiments and blots repeated at least twice.

To realize a bioassay for detection of protein phosphorylation we first used an in vitro cell model to test the antibodies that will be used to construct the biosensor. The cell model was A453, a human skin cancer cell line. This cell can be grown in the lab using specific media and instrumentation. We kept cells in (Dulbecco-Modified Egle Medium) DMEM supplemented with 10% (Fetal Bovine Serum) FBS, and antibiotics. At the time of the experiment 2 identical flask (75cm2) where seeded with roughly 2\*106 cells in 18 ml of DMEM containing FBS and antibiotics. Both flasks were incubated for 3 days at 37°C and 5% CO2. After 3 days both flasks were washed 3 times with (Phosphate-buffered saline) PBS solution in order to remove dead cells, debris and previous medium components.

Subsequently, in order to reduce as much as possible protein phosphorylation at global level, in one flask it was added only DMEM with antibiotics and no FBS (FBS starvation), whereas the second flask was kept in normal condition (DMEM+FBS+antibiotics). Both flasks were incubated for another day at 37°C and 5% CO2, and then proteins were extracted from both flasks.

FBS contains growth factors, hormones, cytokines and other active molecules that stimulate cell growth. Such molecules usually bind extra- or transmembrane receptors that, upon activation, start signaling cascades from the plasma membrane to the nucleus. Basically, such signaling cascades are sustained by kinase proteins that phosphorylate other target proteins (in some cases kinases as well).

Therefore, the absence of FBS in the medium causes a generalized reduction of phosphorylation activity of kinases and consequently a reduction of phosphorylated proteins.

To verify the reduction of protein phosphorylation we tested the phosphorylation status of a marker protein, ERK. Such protein is usually phosphorylated when cells grow at high rate, therefore is a good candidate to verify if the FBS starvation caused any reduction in kinase activity.

We used western blot (WB) assay to test the phosphorylation status of ERK. To perform the test 2 different antibodies were used, one recognizing ERK protein independently by its phosphorylation status, and one that specifically recognizes the phosphorylated form of ERK (pERK).

The WB assay was conducted as following:

* 20 micrograms of total proteins extracted from cells grown in DMEM+FBS+antibiotics were loaded in two consecutive WB wells. These wells will be the control sample for phosphorylation
* 20 microgram of total proteins extract from cells grew in DMEM+antibiotics were loaded in two consecutive WB wells next to the control wells

Two gels with these arrangements were run at the same time and subsequently transferred on nitrocellulose membranes resulting in 2 membranes with 4 protein strips.

At this point one membrane was incubated with the antibody recognizing ERK native protein (independently from phosphorylation), and the second gel was incubated with the antibody recognizing the pERK.

Since the quantity of protein is equal in each gel the signal from ERK should be the same in all wells of both gels, whereas the signal from pERK should be strong in cells grown in normal condition and absent or very poor in cells grown in FBS starved condition.

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To test the ability and sensitivity of our antibodies used in the “Hyperion” we had to perform a Western blot Analysis.

We used two types of antibodies, one antibody that recognises the kinase ERK and one that binds the ERK phosphorylated form pERK.

![brochure](https://static.igem.wiki/teams/4639/wiki/resultserk.png)

As shown above there is one expected band at ~40 kDa when using the ERK antibody (left). When using the pERK antibodies we would expect two bands, one at ~40 kDa and one at ~42 kDa.

The results confirmed the previous as shown at the right picture.

This verified that the antibodies were suitable to use on our biosensor, given that they bind the right protein.

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