iGEM 2022 — Team elte

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4375/wiki/titles/notebook-title-01-min.png)

# February

*We met with the idea of iGEM for the first time in late February, and our journey began immediately.*

### Design

Our Team drafted the first ideas back then, and we were fascinated by iGEM projects where engineered bacteria could sense metabolites and signal molecules and draw action upon them, aiming for a therapeutic effect. To our surprise, we could not find systems triggered by protein epitopes, limiting their applicability.

In addition, we found exciting projects that used signaling pathways involving Nanobodies as sensing molecules (Example: [https://2018.igem.org/Team:NTHU\_Formosa#0](https://2018.igem.org/Team%3ANTHU_Formosa#0)). Such systems are ideal for eukaryotic cells due to their lack of cell wall, but bacteria could harness them by only sensing soluble targets.

During this period, we continuously brainstormed about a signal pathway implemented in bacteria to sense protein antigens.

*That was a magical time, with ideas and late-night discussions instead of preparing for the spring final exams.*

---

# March - April

*Participation in iGEM was pretty unprecedented in our country at the collegiate level, so the competition was unknown to most of our teachers. Despite this, we decided to reach out to supporters, and in **Imre Kacskovics (DVM., Ph.D., D.SC.)**, we found a listening ear. He embraced our initiative and has supported us relentlessly since then.*

*Later we had the chance to present our idea to **Erik Bogsch Jr. (Ph.D.)**, the Head of the Biotechnology Business Unit in Gedeon Richter Plc., the leading pharmaceutical company in our region.*

*With their help and support, our participation has become a reality for which we are highly grateful.*

### Design

 & Human practices

The project idea we pitched to them was plain and simple. We imagined an engineered bacterium sensing external signals coming from tumor tissue and releasing Nanobodies into its environment, decelerating tumor progression. As the registration for the competition has begun, we further improved our idea.

We have decided that our bacteria will target protein antigens and receptor molecules overexpressed on cancer cells. But we have realized this interaction cannot initialize protein expression… **But it can be used as a diagnostic module!** This idea brought a theranostic approach to our project.

After discussions with **Professor Nyitray (Ph.D.)**, we choose Nanobodies as targeting molecules, displaying them on the outer surface of our bacteria. These bacteria-target cell interactions can be visualized via fluorescence. In addition, **Professor Kacskovics** emphasized that we have to choose targets carefully since the main issue with bacterial therapies is their immunogenicity. He advised us to tackle diseases where the bacteria don't need to penetrate the mucosal membrane.

After that, we explored possible ways to give therapeutic features to our project. We have planned to release cytotoxic proteins by sensing tumor microenvironments, such as high levels of lactic acid, low pH, lack of oxygen, or increased body temperature. Talking again with **Professor Nyitray**, we got the idea this may not be the right way because of two things: the targeted molecules are present in healthy tissues too, and also, these environmental conditions are not exclusive. Therefore, such a system would be like a double-edged sword: besides therapeutic effect, it would have certain leakage, and as a consequence harm healthy tissues and have side effects by design.

We have decided to use an external trigger to induce cytotoxic protein release, which can be spatially controlled, so we can mainly limit its effect in the detected area. We chose light because of its non-invasive nature.

This is how NanoBlade was born, the targeted, light-inducible tumor therapy.

We spent the last days of spring designing constructs fulfilling this aim:

*J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA, Intimin-EGFR-sfGFP (InEG), and Intimin-CEA-sfGFP (InCE).*

*The destination vectors were pEV and pET-28a. We have chosen these to have distinct selection markers for the two constructs since our goal was to co-transform either J23101-Intimin-EGFR-J23101-sfGFP or J23101-Intimin-CEA-J23101-sfGFP and J23101-BLADE-ClyA.*

### Safety

The team toured the lab with **Dr. Boglárka Schilling-Tóth** and learned about safety and waste collection rules. We also learned the exact location of the tools and materials so that the lab work could run smoothly in the future. We met with our other Lab Advisor, **Borbála Tihanyi**, and discussed our project and plans.

### WetLab

Participants: Zsóka Csorba, Laura Dénes, Nikolett Emődi, Adrienn Bíró

While we waited for the arrival of the previously ordered sequences from IDT, we started the preparation of the vectors and the materials (LB-media, LB agar). Once the ordered sequences arrived, we began the digestion, ligation, and transformation protocols.

### Hardware

Participants: Fanni Englert, Nikolett Emődi

In the hardware section, first, we have summarized our requirements for the device. With these requirements in mind, we reviewed the literature and the devices which are available commercially. We found that no instrument fit all the requirements, so we started to design our hardware, following the advice of **Tamás Englert**.

---

# May 30 - June 2

### Build

**DNA work**

Participants: Ágnes Golarits, Dóra Kapui, Laura Dénes, Adrienn Bíró

* Transformation of pEV and pET28a vectors into competent DH5α cells.
* The cells grew overnight (ON hereafter) and were isolated with the Miniprep protocol.
* The plasmids were digested with selected restriction enzymes ON.
* The digested vectors were isolated from agarose gel according to the protocol.
* pEV vector was properly digested, this was stored at -20°C until further use.
* pET28a was degraded, in this case, we repeated the preceding steps.

### Human Practices

We consulted with **Márton Bojtár (Ph.D.)**, who is a researcher in “Lendület” Chemical Biology Research Group of the Research Centre for Natural Sciences. His field of research is fluorescent signaling and photoactivatable compounds, so he provided us with useful advice regarding our project.

---

# June 18

### Hardware

Participant: Fanni Englert

The first illumination device was assembled for testing bacterial constructs in Erlenmeyer flasks, Petri dishes, or test tubes.

---

# June 24 - 29

### Build

**DNA work**

Participants: Ágnes Golarits, Dóra Kapui, Laura Dénes

* The sequences ordered from IDT arrived.
* We digested the fragments according to protocol and stored them at -20°C.
* Following standard 3:1 ratio ligation, we transformed the constructs into DH5α cells.
* Colonies were present on the Petri dishes, so we inoculated 4-4 of them to LB-media with ampicillin.
* Plasmids were isolated, and diagnostic digestion was performed on them.

### Wiki

Participant: Nikolett Emődi

The official message has arrived that the teams' Wiki pages are ready for editing. This is the start of months of code-breaking!

---

# July 4 - 5

### Build

**DNA work**

Participants: Dóra Kapui, Laura Dénes, Adrienn Bíró

* BL21 transformation with J23101-BLADE-mCherry and J23101-BLADE-ClyA and J23101-ClyA.

---

# July 6 - 8

### Test

Participants: Zsóka Csorba Dóra Kapui, Laura Dénes, Adrienn Bíró, Nikolett Emődi

We aimed to test the light-inducible protein expression, by monitoring mCherry production via fluorescence (J23101-BLADE-mCherry), and demonstrate ClyA expression with SDS-PAGE. Unfortunately, we get no result since our plasmids did not contain inserts.

### Hardware

Participant: Fanni Englert

Estimated costs and started ordering the parts to build our hardware.

### Graphic design

Participants: Nikolett Emődi, Fanni Englert

Our official logo is now available! It was quickly put on our team T-shirts, stickers, and badges to promote our team.

---

# July 9 - 10

### Build

**DNA work**

Participants: Ágnes Golarits, Dóra Kapui, Laura Dénes, Adrienn Bíró

* The J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA constructs were found to be inappropriate during digestion.
* Amplification by PCR of J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA from the IDT sequences as templates.
* Digestion was repeated under the same circumstances, ligation with a 5:1 ratio, and transformed.
* No colonies had grown.
* We concluded that the ligation was inappropriate.

---

# July 11 - 14

### Build

**DNA work**

Participants: Laura Dénes, Nikolett Emődi

* BL21 transformation with J23101-BLADE-mCherry and J23101-BLADE-ClyA and J23101-ClyA
* Plasmid isolation from BL21
* Diagnostic digestion

We had no insert.

### Test

Participants: Zsóka Csorba Dóra Kapui, Laura Dénes, Emődi Nikolett, Adrienn Bíró

We aimed once again to test our constructs:

* Blue light incubation OV* mCherry measurement* Detect the production of ClyA protein from cells using SDS-PAGE

Since we did not observe the appearance of proteins during the cultivation, we performed control digestion on the plasmid we were working with (this was necessary because the sequencing results were not yet available). This revealed that the plasmid did not contain the insert.

### Design

Note: After concluding that our ligation attempts had failed twice, we began a troubleshooting series to obtain our results. During this, we went through all the possibilities that could have caused our inefficient cloning (competent cell, vector, or insert not digested properly, failed ligation). This season started in mid-July and ended at the end of August.

---

# July 14 - 22

### Build

**DNA work**

Participants: Laura Dénes, Adrienn Bíró, Ágnes Golarits

* J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA, J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA were restarted with digestion and ligation.
* A new batch of DH5α competent cells was made.
* pETARA vector digestion (XbaI, XhoI), isolation from agarose gel (Picture 1).

![](https://static.igem.wiki/teams/4375/wiki/notebook/notebook1fin-01-min.png)

No colonies were obtained.

### Design

Note: Since we suspected that there might be a problem with digestion since we have changed the cells, we chose a new vector and ordered primers to have more nucleotides in front of the cleavage site and thus have more efficient digestion. You can find the [primers](https://2022.igem.wiki/elte/parts) we used as: BBa\_K4375022, BBa\_K4375023, BBa\_K4375024, BBa\_K4375025, BBa\_K4375026, BBa\_K4375027, BBa\_K4375028). Our newly chosen plasmid was pETARA. The reason behind our choice was that once pETARA is digested, the excised fragment is visible on agarose gel, giving us proof that plasmid digestion was successful.

### Collaboration

****07.19. - DTU iGEM meeting****

On this day we met our mentor team, where they gave us a fantastic presentation on the essential aspects of iGEM and gave us a new dose of energy to continue our work.

**07.20. - UPenn meeting**

During the meeting with the UPenn team, we got to know each other and each other's projects. Here we planned in advance to try to carry out a joint measurement during the competition.

---

# July 25 - 31

### Build

**DNA work**

Participants: Laura Dénes, Ágnes Golarits

* PCR amplification of J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA, J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA.
* Isolation of products, digestion of J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA with XbaI, XhoI, of J23101-sfGFP with XbaI, PaeI, of J23101-Intimin-EGFR, and J23101-Intimin-CEA with XhoI, PaeI.
* Ligation with the previously tried 3:1, 5:1 ratio, and a new 10:1.

No colonies were obtained.

### Design

Note: We consulted with our Lab Adviser **Boglárka Schilling-Tóth** to try to find the problem. Since neither We, neither She could find a problem She advised us to try it again in another lab, a change of scenery will help us. We choose the three most important constructs:
J23101-BLADE-mCherry, J23101-BLADE-ClyA, and J23101-ClyA, and asked the Vascular Research Group to use their lab.

* Transfer of primers, J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA DNA templates, and XhoI, XbaI digested pETARA plasmids to Vascular Research Group Lab. The transfer was made in an ice-filled box.
* PCR of J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA.
* Agarose gel electrophoresis and isolation of results.
* Digestion of J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA with XbaI, XhoI. Direct digestion according to protocol.
* Ligation, with a 3:1 ratio.
* Transformation to Xl1Blu competent cells.
* Colonies had grown. 4-4 were selected and inoculated into LB-medium, 1000x ampicillin.
* Miniprep isolation of samples.
* Diagnostic digestion of J23101-BLADE-mCherry, J23101-BLADE-ClyA, J23101-ClyA DNA clones.
* Separation of digested fragments with agarose gel electrophoresis.
* Positive plasmid constructs were obtained (Picture 2 and 3).
* Positive samples were sequenced.

![](https://static.igem.wiki/teams/4375/wiki/notebook/note23-01-min.png)

By the end of July, the cloning of J23101-BLADE-ClyA, J23101-BLADE-mCherry, and J23101-ClyA were finished and ready to be used.

### Collaboration

**07.31. - Worldshaper-HZ**

We met the Chinese team and once again had a fantastic meeting.

### Graphic design

Participants: Nikolett Emődi, Laura Király

Our logo appeared on the team's official Instagram page, and we also uploaded our first post introducing the iGEM competition to our followers.

---

# August

### Build

**DNA work**

As August began, we started to work with our finished constructs and at the same time continuing our efforts to finish the J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA constructs.

We transformed the resulting good J23101-BLADE-mCherry, BLADE-CylA, and CylA constructs into BL12-DE3 cells.

### Human practices

We conducted a survey to find out the perception including the most common fears and misconceptions about GMOs and their medical uses. All of this has allowed us to specifically address these concerns in our project implementation and education activities.

---

# August 1 - 12

### Build

**DNA work**

Participants: Laura Dénes, Adrienn Bíró

Note: since we were sure that the pETARA was digested and there was nothing particular to imply that the J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA inserts were incompletely digested we began a new troubleshooting season.

* Digested J23101-sfGFP, J23101-Intimin-EGFR, and J23101-Intimin-CEA and pETARA were ligated with T4 and Hi-T4 ligases simultaneously. Ligation ratio was 1:1, 1:2, 1:5.
* Transformation was done into DH5α competent cells.

No colonies had grown.

* Repeat protocol into Xl1Blu competent cells.
* Change to carbenicillin.

No colonies had grown.

Note: since we suspected there might be some issue with ampicillin, we changed it to carbenicillin.

**In vitro experiments**

Participants: Laura Király, Zsóka Csorba

* We thawed A431 cells.
* As a preliminary experiment for our cytotoxicity measurements, we optimized the ideal cell number to be plated.

### Test

Participants: Laura Király, Zsóka Csorba

We carried out our second attempt to characterize light-dependent ClyA production using SDS-PAGE. Unfortunately, we had some problems with gel casting.

### Hardware

Participant: Fanni Englert

* Explored the software for programming illumination, and tested with individual LED strips.
* Beginning of making the unique illumination area and soldering the LEDs.

---

# August 15 - 18

### Build

**DNA work**

Participants: Laura Dénes, Ágnes Golarits

* Performed PCR on the J23101-Intimin-EGFR and J23101-sfGFP constructs.
* Ligation followed in 3:1 ratio and transformation into Xl1Blu competent cells.
* Colonies had appeared on the Petri dishes.
* Inoculated to LB-media.
* Plasmid preparation.
* Diagnostic digestion.

Note: The diagnostic digestion yielded strange degraded plasmids. Additional colonies were inoculated from the plate. Again we got the degraded DNA.

### Test

Participants: Laura Király, Zsóka Csorba

We carried out our third yet first successful attempt to characterize light-dependent ClyA production using SDS-PAGE.

### Collaboration

**08.18. - Wageningen\_UR**

We met the Wageningen team's project idea, and they presented us with their great questionnaire. Here we decided to evaluate the questionnaire together to improve the Human Practice part of the project.

### Hardware

Participant: Fanni Englert

* The individual illumination area for the 96-hole plate is prepared.
* Unfortunately, it was found that in 3 of the 12 columns, not all the LEDs lighted up.
* It was discovered that this could be caused by a factory defect that LEDs have inadequate plastic cladding. Therefore, a short circuit occurs when they come into contact with the aluminum plate, which is responsible for heat dissipation.
* Ordered the components needed for the row-by-row programmable unique illumination area.

### Wiki

Participant: Nikolett Emődi

Our opening page is up on Wiki! In the meantime, we learned the mysteries of HTML and CSS, and with great enthusiasm, we edited the page from sunrise to late at night with the ever-expanding material and knowledge.

---

# August 22 - 26

### Build

**DNA work**

Participants: Laura Dénes, Ágnes Golarits

Note: We again tried at the **Vascular Research Group lab** with our work.

* Where we performed PCR.
* Ligation followed in 3:1 ratio and transformation into Xl1Blu cells.
* Colonies had appeared on the Petri dishes.
* Colonies were inoculated to LB-media.
* Plasmid preparation.
* Diagnostic diegestion and ligation.
* Colonies have again appeared, but control digestion yielded strange degraded plasmids.

Note: Since the strange smear given by the plasmid was difficult to explain, we decided to check the presence of J23101-Intimin-EGFR and J23101-sfGFP in the sample by PCR.

* PCR to check the presence of J23101-Intimin-EGFR and J23101-sfGFP in the sample.
* Agarose run.

Note: The gel image showed that J23101-sfGFP was present in the sample, and a faint signal was obtained for J23101-Intimin-EGFR. We ran the PCR to sequence the whole insert isolated from the gel.

---

# August 29 - September 2

### Build

**DNA work**

Participants: Laura Dénes, Adrienn Bíró, Ágnes Golarits

- PCR with plasmids J23101-Intimin-EGFR and sfGFP-specific primers.
- Transformation of plasmid into XL1Blue.

No colonies were obtained.

Note: We did not get a satisfactory result, but to have enough samples for further work we transformed the DNA used in the PCR. No colonies were obtained, which implied that the DNA was degraded.

### Hardware

Participant: Fanni Englert

* The row-by-row programmable unique illumination area is ready to use.
* Started to fix the broken column-by-column version.

### Collaboration

**09.02. -NTHU\_Taiwan**

The Taiwanese team contacted us on this day with great collaboration ideas, which we were delighted to join. Within a few days, we sent them the requested form.

### Graphic design

Participant: Nikolett Emődi

Art Work for the iGEM jamboree is ready! In this artwork, we displayed the main elements of our project. We hope to see it in Paris!

---

# September 5 - 11

### Design

As we cannot overcome the obstacles of the two-insert ligation, we have designed a diagnostic construct that consists of a single insert (called MiniNano, to know more, take a look at our  [Parts Page](https://2022.igem.wiki/elte/parts)) and we ordered from IDT. **Thank you, IDT!**

### Hardware

Participant: Fanni Englert

Column-by-column version of our LED-panel was ready to use.

### Test

Participants:Laura Király, Zsóka Csorba, Márton P. Nyiri

We tested our new LED panel for the first time, and it was partially successful.

### Human Practices

Seeing that people are sensitive to the safety issues of genetically engineered bacterial therapies, we interviewed a professional from the Imperial College of London, **Dr. David T Riglar (Ph.D.)**. His lab is constantly trying to develop innovative technologies, such as living-engineered probiotics.

We were lucky to consult **Dr. Árpád Patai**, gastroenterologist, and internist. During his Ph.D., he studied the epigenetics of colorectal cancer and is currently involved in the treatment of various gastroenterological diseases at the 2nd Department of Internal Medicine of Semmelweis University. The conversation with him was beneficial for us, as it highlighted many factors that could greatly influence the future applicability of our project.

### Collaboration

**09.11 - BOKU-Vienna**

On this day, we participated in a modeling webinar organized by the BOKU-Vienna team to help us make our model more accurate.

---

# September 12 - 16

### Test

Participants:Laura Király, Zsóka Csorba

We tested our new LED panel for the second time; it was not a successful attempt.

### Collaboration

**09.15. - MSP-Maastricht**

The MSP-Maastricht team approached us to help distribute their survey, which we are happy to do.

---

# September 19 - 23

### Test

Participants: Zsóka Csorba, Laura Dénes

We aimed to test our constructs on Blood agar, using our J23101-BLADE-ClyA, and for negative control J23101-BLADE-mCherry constructs. It was not a successful attempt, because all the plates dried up during the incubation.

### Collaboration

 & Human practices

**09.22. - Wageningen-UR**

During the meeting, a joint evaluation of the questionnaire was carried out, which helped us to improve the integrated Human Practice.

### Collaboration

**09.23. - Eastern Europe meeting**

On this day, the largest meeting of its kind took place, where we invited three other Eastern European teams for a meetup. See details on the Eastern Europe meetup page.

---

# September 26 - 30

### Test

Participants:Zsóka Csorba, Laura Dénes

Our second attempt to test the light-inducible ClyA hemolytic activity on Blood agar was partially successful.

### Education

Our survey has shown us that people's knowledge about GMOs, not to mention SynBio, is quite limited. Therefore, our Team aimed to broaden their understanding, and we enrolled as presenters at this year’s European Researcher’s Night at ELTE Faculty of Sciences. Our focus was to familiarize the visitors with synthetic biology in general, its applications, and the tools we often use in our work. The participants had the chance to learn about NanoBlade and experience biochemistry and biology's marvels firsthand. Approximately 300 children (and others who were interested) visited our exhibition.

---

# October 1 - 12

### Build

**DNA work**

Participant: Laura Dénes

Since MiniNano arrived this week, we aimed to insert this construct into pETARA plasmid. The arrived construct was digested with XhoI/XbaI, then we ligated them with the vectors. Transformation was done into Xl1Blu competent cells.

Colonies were obtained after the transformation.

### Collaboration

**10.7. - Discussion with Team UPenn**

Team UPenn had troubles with their irradiation experiments, so as part of our collaboration, they sent us their plasmid constructs to try them out with our LED-panel. During this meeting, we discussed our questions about their protocol and how to carry it out.

### Education

Participants: Adrienn Bíró, Laura Király

We visited a high school class where we held an interactive lesson, starting with a Kahoot! quiz on GMOs and their everyday use. After that, we were happy to answer their questions and discuss their views on the topic. We also briefly presented our project and showed them our lighting devices. The students were curious and asked a lot of questions. At the end of the lesson, we played some games, such as plasmid puzzle game.

### Wiki

Participants: Nikolett Emődi, Fanni Englert

In the last few weeks, Wiki has been in a non-stop editing process. The latest measurement results, collaborations, parts, and science popularization elements added to the site. We hope that you will like it too!

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4375/wiki/titles/eng-suc-01-min.png)

# Introduction

During our project, we faced numerous challenges in the engineering part. These obstacles sometimes proved to be hard ones to overcome, but we can say it honestly that we did not step back. In this short review, we present our troubleshooting cycles through which we have learned: biology is challenging, and often the simplest designs can fail. With creativity and rational design, we managed to overcome several barriers standing between us and success.

![](https://static.igem.wiki/teams/4375/wiki/engsucc/eng-succ-min.png)

# BLADE

### 1. Cycle: False positive diagnostic digestion → Improved plasmid isolation

#### Research

In our project, the cytotoxic protein needed to be produced only in response to an external stimulus, in our case, blue light. The system is based on a promoter that can be regulated by a protein that dimerizes in response to blue light, called BLADE and described by Romano et al. [1]

We planned to test this design in the BL21 strain and characterize it by illuminating the bacteria with blue light and examining protein production.

To facilitate this, we designed not only the CylA protein of our choice but also a model construct using a reporter protein, fluorescent mCherry, behind the promoter. This, we could check the promoter's function by measuring the fluorescence.

#### Imagine

This construct was designed to be cloned into a pEV vector using conventional cloning techniques. For this purpose, a bacterial codon-optimized sequence was ordered from IDT using XbaI and XhoI enzyme cleavage sites interspersed.

#### Design

We decided to commission the synthesis of the BLADE to construct the downstream protein coding region from IDT in one step due to the short time available and to minimize the upstream risks. Thus, with a simple single-incision ligation, our plasmid can be created. In addition, several members of the team had extensive experience in this cloning technique.

#### Build

For cloning, we digested the vector and the ordered inserts with the appropriate restriction enzymes and performed the ligation with T4 ligase.

#### Test

Plasmids were isolated and digested with XbaI and XhoI enzymes, and gel electrophoresis was performed on the samples. Although faintly visible on the gel (because we did not digest enough DNA), the insert was visible in some clones. So we selected these and started protein production studies with them. However, no results were obtained. So as a control, we re-isolated the plasmid and digested it, and this showed that the insert was not present in the plasmid, so it was clear why we did not get any results from the protein production assay.

#### Learn

We have learnt that an accurate gel image is essential for a proper conclusion because if we go ahead with an uncertain result, it means a lot of extra work and time for us.

#### Improve

By isolating the plasmid, more attention was paid to obtaining a sample of sufficiently high purity and higher concentration. This was significant because the low plasmid concentration in the first attempt meant that we did not digest enough to see the results properly on the gel.

#### Test

We did not get colonies on the plate.

### 2. Cycle: No colonies after transformation → New vector

#### Research

Behind the lack of colonies, we first suspected that either the inserts or the vector were not digested properly. Digestion of the inserts is difficult to check, as only a few bases with a few overhanging ends are cleaved. In the case of the plasmid, this is easier to check, and we could test the ability of the enzymes used to work.

#### Imagine

In the pEV vector, the XhoI and XbaI cleavage sites are very close, so it is not so clear in the gel image whether it has been digested properly. So we looked for a vector where if we digested with the two enzymes, a clearly visible fragment would fall out.

#### Design

We chose the pETARA vector and planned to digest it for cloning, as a fragment of around 1000 bp is lost from this plasmid during XbaI and XhoI digestion.

#### Build

We have digested the pETARA vector. The gel clearly showed the fragments that had fallen out, so we could be sure of proper digestion. The previously digested inserts were ligated into the vector.

#### Test

We did not get colonies on the plate.

#### Learn

We saw that the vector was digested properly, so the problem is not with the enzymes and not with the vector digestion.

### 3. Cycle: No colonies after transformation → Change of used cloning bacterial strain

#### Research

Since we again did not receive any colonies, we took a look at what the problems might be. It was suggested that there might be a problem with the competence of the competent cells, so we tried to eliminate this.

#### Imagine

Since we found that the DH5α cells were not as competent as Xl1Blue, we switched to this one.

#### Build

We performed the cloning according to the previous protocol with a freshly digested vector, but in the end, the transformation was performed in Xl1Blue cells.

#### Test

We did not get colonies on the plate.

#### Learn

From this, we learned that the problem may not be with the competence of the competent cells. (Also, competent cells from the same stock have been used by others and worked well for them.)

### 4. Cycle: No colonies after transformation → Introducing extra nucleotides to the close of the DNA fragments

#### Research

After several failed transformations, we checked the sequences again and asked our mentors what the problem might be. They pointed out that we hadn't left enough nucleotides at the beginning of the cleavage sites, so it was probably due to the low efficiency of digestion of the insert.

#### Imagine

To solve this problem, we had to introduce extra nucleotides in front of the cleavage sites in the sequences.

#### Build

We designed primers that contained extra nucleotides beyond the cleavage sites, allowing us to use PCR to extend the sequence of the inserts and thus increase their digestion efficiency.

You can find these [primers](https://2022.igem.wiki/elte/parts) in the Registry as Forward primer for BLADE (BBa\_K4375024), Reverse primer for J23101-BLADE-ClyA (BBa\_K4375025), and Reverse primer for J23101-BLADE-mCherry (BBa\_K4375026).

#### Test

After the PCR reaction, a product of the correct size was obtained, purified from gel digest, and ligated into a freshly digested pETARA vector. However, by the next day, we still had no colonies on the plate. We set the ligation reaction aside and ran it to see if we could see any occluded vectors. This gel pattern was as expected.

#### Learn

At this point, we had tested everything we thought might be causing the problem (enzymes, inadequate digestion of vector or inoculum, competent cells not functioning properly, antibiotic degradation). So, in consultation with our advisor, we took it to another lab to see if we could get a hit using the tools they had.

### 5. Cycle: No colonies after transformation → New lab

#### Build

We took all the samples to the Vascular Research Group's lab and went through the entire process again, from PCR amplification of the inserts to starting the work.

#### Test

Here we obtained colonies, and after plasmid isolation, we performed diagnostic digestion. The gel electrophoresis confirmed the presence of the correct insert in the plasmid, and the presence of the fluorescent protein was detected.
So after a long time, we finally achieved success.

#### **Test+1**

As a good researcher, he did not leave us alone as the reason for our failure. It was suspected that perhaps one of the batches of LB agar was not good. To check this, we spotted competent cells transformed with a plasmid containing the appropriate resistance on one of the plates. Since no colonies were found here, we concluded that the failed agar might indeed be the cause of our ordeal.

# Hardware

### 1. Cycle: How to test the bacterial construction as early as possible → Modular illumination device consisting of home lighting elements

#### Research

The BLADE expression system is blue light-inducible (~450 nm) therefore an appropriate irradiation setup is needed for testing our constructs.

#### Imagine

We have listed our requirements for the irradiation setup: (1) the LED must emit light at the optimal wavelength, (2) it can be cooled, (3) it can be put into an incubator, (4) it needs to have a dimmer function, and (5) it must be safe. To test our bacterial construction as soon as possible, we first thought about a device that would be quick to assemble and easy to use.

#### Build

We have built our first irradiation setup with components that can be found in a household and easily obtainable in a shop. The unique illuminating area is a plastic cylinder, the blue LED strip is attached to the wall which is cooled by a PC fan.

#### Test

The illumination device was successfully installed and shaken in the incubator. During the experiment, the samples did not overheat, so air cooling was suitable. The first test reactions showed that the wavelength was appropriate, and the experiment was successful.

#### Learn

This device was adequate to draw significant conclusions about how our system works. We understood that it was not suitable for more complex experiments or testing its interaction with mammalian cells.

### 2. Cycle: How to carry out mammalian cell illumination experiments → Creating RaccOpto

#### Research

From the drawbacks of the previous hardware, we knew that our illumination device had to be programmable. Furthermore, we needed multiple channels if we wanted to illuminate a 96-well plate so that we could test it with different illumination programs per row or column.

#### Imagine

Since the literature and commercially available devices did not meet all our requirements, we wanted to assemble our lighting device from modular parts that could be bought in stores. We aimed to make installation and repair as easy as possible.

#### Build

As we found suitable modular units for each of the hardware tasks, our main task during the construction was to create a unique lighting area. To dissipate the heat, we placed the blue LEDs in 96 holes drilled in a 6 mm thick aluminum sheet, cooled by a PC fan.

#### Test

Fearing hardware incompatibility of some parts, after assembly, we were pleased to find that our system worked correctly and programming was successful. Unfortunately, we found some columns did not have all LEDs lighted up.

#### Learn

Finding the fault, we discovered that some LEDs have an incorrect plastic coating due to a factory defect and become short-circuiting when in contact with the aluminum plate. Paying close attention to this, we corrected the illumination area and created a per-pole controllable device in addition to a per-row controllable one.

---

[1] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. *Nat. Chem. Biol.* **2021**, *17*, 817–827. <https://doi.org/10.1038/s41589-021-00787-6>.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4375/wiki/titles/experiments-title-01-min.png)

# How to make LB medium?

Recipe for 500 ml LB medium:

* 5 g Tripton
* 2.5 g NaCl
* 2.5 g Yeast extract
* 500 mL MQ water
* glass bottle

Steps:

After you have measured the ingredients, mix the solution with a magnetic stirrer approx. 10 minutes or until the components dissolve.
You should sterilize them in an autoclave and after they have cooled down they are ready to use.
Add the right antibiotics to the medium in 1:1000 dilution.

---

# How to pour agar plates?

* Prepare your LB agar medium.
* If you autoclaved it freshly please wait until it cooled down enough to handle. (<60°C)
* If you work with a previously prepared medium, you must warm it up with a microwave oven.

+ Please loosen the cap of the bottle.
+ Warm it for about 1 minute, and shake it vigorously.
+ Then continue the warming up but with close attention. You should mix it every 20-30 seconds or if you see intense bubbling.
+ You can stop when the solution is clear and you do not see any gel floating.
+ You have to wait until it's cool enough to handle with your bare hands.

* From this step continue your work in a sterile environment!

+ Place your plates in a laminar box.
+ When you are opening your bottle, sterilize the opening with flame.
+ Add antibiotics at a 1:1000 ratio.
+ Pour as fast as possible approx. 8-10 mL medium into the plate, close it, and mix it with a circular motion.
+ Wait for 20 minutes to solidify.
+ Store the plates at 4°C.

---

# Insert restriction digestion and purification

* Weigh the reaction mix in an Eppendorf:

+ Add the required enzymes, the DNA template, and the right buffer (buffers were chosen according to Thermo’s webpage, [DoubleDigest](https://www.thermofisher.com/hu/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html)).
+ Restriction enzymes used for construct digestion:

- J23101-BLADE-mCherry: XbaI, XhoI
- J23101-BLADE-ClyA: XbaI, XhoI
- J23101-ClyA: XbaI, XhoI
- J23101-Intimin-EGFR: XbaI, PaeI
- J23101-Intimin-CEA: XbaI, PaeI
- J23101-sfGFP: XhoI, PaeI

* Put it in a 37°C incubator for one hour or overnight.
* Purification according to the kit protocol.

We performed DNA purification with a nucleic acid purification kit (Thermo Fischer Scientific: GeneJET PCR Purification Kit;<https://www.thermofisher.com/order/catalog/product/K0701>) according to the manufacturer's protocol (differences: elution volume: 25 µL).

---

# Plasmid digestion

* Add the required enzymes, the DNA template, and the buffer.
* Restriction enzymes used for plasmid digestion:

+ pET28a: XbaI, XhoI
+ pEV: XbaI, XhoI
+ pETARA: XbaI, XhoI

* Put it in a 37°C incubator for one hour or overnight.
* Mix the reactions with 6x Purple Loading Dye.
* Add Generuler 1 kbp Plus Marker. (Thermo: <https://www.thermofisher.com/order/catalog/product/SM1331>)
* Run agarose gel electrophoresis, 100 mV, 45 min.
* Cut out the band of digested plasmid with a sterile scalpel.
* Purification according to the kit protocol. (Thermo Fischer Scientific: GeneJET Agarose Purification Kit: <https://www.thermofisher.com/order/catalog/product/K0691>)

---

# Ligation

Ligation was done in several different ratios: 1:1, 1:2, 1:3, 1:5 and 1:10.

* Calculate how many µL of insert and vector you need to introduce to the reaction mix based on the equitation below:
  *required mass insert (g) = desired insert/vector molar ratio x mass of vector (g) x ratio of insert to vector lengths*
* Add T4 ligase, 10x Ligase buffer, sterile distilled water, and DNA components in appropriate proportions.
* Incubate at room temperature for 1 hour.

*For Hi-T4 ligase reaction, the following protocol was recommended:

* Add 10x T4 DNA Ligase reaction buffer, calculated insert and vector ng-s, sterile distilled water, Hi-T4 ligase.
* Incubation for 10 minutes at 37°C, heat inactivation for 10 minutes.*

---

# Transformation

The plasmid vectors were cloned in *E. coli* DH5α and Xl1Blu competent cells.

* Competent cell name: **DH5α**
* Competent cell volume: 100 µL
* Plasmid vector volume: 1 µL
* Mix the plasmid with cells
* Incubation on ice: 1 min
* Heat shock: temperature: 42°C, time: 2 min
* Incubation on ice: 5 min
* LB medium volume: 0.1 mL add to transformation mix
* LB + Antibiotic plate: Pipette the cells onto the agar and then spread them out with the burnt and cooled metal stick. Smear the cells until they are absorbed by the agar. Seal the edge of the plate with cork film and place it in the incubator.
* Incubation: temperature 37°C, time: overnight

* Competent cell name: **Xl1Blue**
* Competent cell volume: 40 µL
* Plasmid vector volume: 1 µL
* KCM mix: 8 µL
* Sterile distilled water: 27 µL
* Mix the plasmid with cells
* Incubation on ice: 20 min
* Heat shock: room temperature, 10 min
* LB medium volume: 0.1 mL add to transformation mix, shake at 180 rpm at 37°C, 1 hour.
* LB + Antibiotic plate: Pipette the cells onto the agar and then spread them out with the burnt and cooled metal stick. Smear the cells until they are absorbed by the agar. Seal the edge of the plate with cork film and place it in the incubator.
* Incubation: temperature 37°C, time: overnight

---

# Growing bacteria

* Pick up colonies with a pipette tip and put them in 5 mL of LB-medium with 1000x antibiotics (ampicillin/carbenicillin)
* Then incubate at 37°C O/V, shaking at 180 rpm.

---

# Miniprep

Isolation of plasmid from overnight culture according to the kit protocol (we selected Thermo Fischer Scientific:GeneJET Plasmid Miniprep Kit: <https://www.thermofisher.com/order/catalog/product/K0502>).
After the purification was measured the concentration with NanoDrop.

---

# Diagnostic Digestion

* Weigh the reaction mix in an Eppendorf.

+ Add the required enzymes, the DNA template, and the selected buffer (for each enzyme pair we selected the appropriate buffers according to Thermo Fisher  [DoubleDigest](https://www.thermofisher.com/hu/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html) web page).

* Incubate at 37°C for 1 hour.
* Mix the reactions with 6x Purple Dye.
* Add Generuler 1 kbp Plus Marker. (Thermo, <https://www.thermofisher.com/order/catalog/product/SM1331>)
* Run agarose gel electrophoresis, 100 mV, 45 min.

---

# Gel run

* Measure 0.5 g agarose on the scale, pour it into a flask and fill it with 50 mL of 1x TAE buffer.
* This mixture is heated in a microwave oven to obtain a syrupy liquid without translucent agarose particles (this can be achieved by shaking the jar frequently while wearing protective gloves).
* When the solution is ready, cool the flask to hand temperature and add EcoSafe 2.5 µL (from 4°C) of dye to the agarose.
* The mixture is poured into the bath and any bubbles that may form are to be destroyed with a pipette tip.
* Leave to solidify for at least 20 minutes. After the gel has solidified, the vat is filled with 1x TAE buffer to cover the gel by half a cm.
* Run gel electrophoresis at 100 mV, for 45 min.

---

# BL21 transformation

For one construction:

* 40 µL BL21 competent cells
* 8 µL KCM mix (calcium and magnesium ion mix)
* 27 µL sterile distilled water
* 1 µL plasmid
* 20 min on ice
* 10 min room temperature
* Add 100 µL LB-medium.
* Shake on ~180 RPM, 37°C, for at least 1 hour.
* Add the mix to LB-medium for starter culture (usually 50 mL).
* Add 1:1000 antibiotics.

---

# Making competent cells

Storage solutions should be stored in sterile containers at 4°C. 100 mL should be made at a time.

TFB solution I: pH= 5.8 (adjust very carefully with 10% acetic acid)

* 100 mM RbCl
* 50 mM MnC
* 30 mM Potassium acetate
* 10 mM CaCl2
* 15 % glycerol

TFB solution II: pH=6.8 (adjust carefully with KOH)

* 10 mM MOPS
* 10 mM RbCl
* 75 mM CaCl2
* 15 % glycerol

Pre-culture:

* Dip sterile tip (under sterile booth) into frozen BL21 cells (-70°C).
* Make 5 mL LB with selection antibiotics + tip with the cells (pipette tip should also be placed in the vial).
* Keep it in a shaker overnight at 37°C, 200 rpm.

Culture:

* Make a 50 mL LB culture medium with antibiotics from 500 µL BL21 (this is the pre-culture grown the night before).
* Keep the cultures for 120 minutes at 37°C (afterward 1 mL sample should be taken at intervals, optical density is measured at 600 nm when OD is between 0.3-0.6 (≈0.4), and the solution can be removed from the shaker.
* Transfer 50 mL to a sterile falcon and centrifuge at 3600 rpm, 20 minutes at 4°C.
* Discard supernatant.
* Resuspend the cells in 10 mL TFB I.
* Keep it for 20 minutes on ice.
* Keep it for 20 minutes at 3600 rpm, 4°C.
* Discard supernatant.
* Resuspend the cells in 1 mL TFB II.
* Keep it for 20 minutes on ice.

Freezing:

* After the addition of 15 % sterile filtered glycerol (regardless of whether competent preparation solutions already contain glycerol).
* Put in it 1 mL 350 µL 50% glycerol.
* Put 80-100 µL from it into Eppendorf tubes
* Store at 80°C.

---

# Cell culturing

Materials:

* Complete medium

+ Serum-free DMEM (Dulbecco’s Modified Eagle Medium, Gibco, Thermo Fisher Scientific, Waltham, MA, USA)
+ 10 % FBS
+ 5 mM glutamine
+ 1 % penicillin-streptomycin

* PBS
* Trypsin
* Resasurin (Thermo Fischer)
* Sterile Flasks (T-25, T-75)
* Serologic pipettes

---

# Thawing cells

* Frozen cells are stored in liquid nitrogen until immediate use. When removing from liquid nitrogen, place vials on dry ice for a few minutes, or start thawing immediately.
* The amount in 1 cryovial (1 mL) is sufficient to start a culture in a 25 cm2 flask.

Work quickly and carefully.

---

# Prepare

* Complete medium pre-heated in an incubator (37°C, 5 % CO2) in the flask to be used for culturing (25 cm2 flask with 5-6 mL cell culture media). It is important to avoid excessive alkalinity of the medium during the recovery of the cells. It is suggested that, before the addition of the vial contents, the culture vessel containing the complete growth medium be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
* Complete medium pre-heated
* Pre-heated water bath (37°C)
* 5 mL pipettes (sprayed with EtOH)
* 15 mL tubes (sprayed with EtOH)

---

# Steps

* Remove the vial of frozen cells from liquid nitrogen and quickly thaw in a 37°C water bath by gentle agitation (it should take between 1 and 2 min). To reduce the possibility of contamination, keep the O-ring and cap out of the water. Remove from the water bath when there is only a small ice chunk inside the vial (it will thaw by the time you start the process).
* Just before the cells are completely thawed, decontaminate the outside of the vial with 70 % EtOH and transfer the cells to a sterile 15 mL tube.
* Add 9 mL of pre-heated medium to the cells. Start very carefully dropwise then you can add faster.
* Centrifuge the cells at 500 x g for 5 minutes at room temperature.
* Remove the medium using a pipette very carefully.
* Resuspend the cell pellet in the pre-heated media for the cell culture flask.
* Place the cell suspension into the cell culture flask and place it in an incubator (37°C, 5 % CO2).
* Incubate overnight or for two days.

Note: Wait for a few passages (2-3) until cells are fully recovered before starting to use them.

---

# Subculturing

* Passage cells when they are 80 % confluent.
* Passage cells 2-3 times a week.

ALWAYS label the flask: the date, your name, passage number, cell type, and subculturing ratio.

* Remove supernatant with a pipettor.
* Pipette 5 mL of preheated PBS, and wash out the remnant medium. Wash the cells gently but thoroughly.
* Trypsinize cells with 1 mL 0.25 % trypsin solution in the case of the T25 flask (in the case of T75 use 2 mL).

+ Incubate A431 cells for 15 min at 37°C, 5 % CO2.
+ Incubate HTC116 cells for 2-3 min at 37°C, 5 % CO2.

* In the meantime prepare a flask with 4 mL media and incubate it for at least 15 min at 37°C, with 5 % CO2.
* Add 2 mL of pre-heated media to stop the enzyme reactions.
* Hit gently the bottom of the flask a few times.
* Check the detachment of the cells.
* Suspend cells with a pipettor, and place the cell suspension into a clean Falcon tube.
* Centrifuge cells for 5 min at 500 g.
* Remove supernatant and resuspend cells in 5 mL of pre-heated media.
* Add 1 mL cell suspension to the pre-heated flask.
* Check cell density.

---

# Cell counting

* Clean the chamber and cover the slip with alcohol. Dry and fix the coverslip in position.
* Harvest the cells.
* Add 10 μL of the cells to the hemacytometer. Do not overfill.
* Place the chamber in the inverted microscope under a 10X objective.
* Count the cells in the large, central gridded square (1 mm2). The gridded square is circled in the graphic below. Multiply by 10^4 to estimate the number of cells per mL. Prepare duplicate samples and average the count.

---

# Cell freezing

Freeze cells at a density of at least 1 x 10^6 cells/mL (total volume in 1 vial: 1 mL)
(A T-75 flask usually yields 3-4 cryovials.)

Prepare:

* Ice bucket
* Labeled cryovials (2 mL)
* Mr. Frosty (filled with IPA and pre-chilled on ice or a few minutes at -80°C)
* Liquid nitrogen container for cryovials

Steps:

* Culture the desired quantity of cells to 70-90 % confluency. (T-75 flask).
* Remove all medium from the flask and wash the cells once with 10 mL PBS to remove excess medium and serum. The serum contains inhibitors of trypsin.
* Add 2 mL trypsin solution to the monolayer and incubate the cells for the time given above, until the cells detach.
* Add 10 mL complete medium and transfer the cell suspension to a 15 mL sterile conical tube.
* Count cells.
* Place the right amount of cell suspension into the cryovial.
* Total it for 0,9 mL with media.
* Supplement the solution with 100 µL DMSO.
* Place the vials (max 18) in Mr. Frosty compartments. Place Mr. Frosty at -80°C for at least 24 h. (Mr. Frosty is a controlled-rate freezing apparatus ensuring a freezing rate of a decrease of 1°C per minute).
* Transfer cryovials to liquid nitrogen storage.

---

# Cell viability assay

* Seed cells into 96-well plate (in antibiotic-free DMEM).
* After 24 h remove the supernatant and add 100 µL 1xPBS and resazurin dye solution in an amount equal to 10 % of 1xPBS volume.
* Return cultures to the incubator for 2 h.
* Read fluorescence at a wavelength of 590 nm using an excitation wavelength of 560 nm (SpectraMax ID3 Multi-Mode Microplate Reader, Molecular Devices).

---

# SDS-PAGE

First day:

* Incubate J23101-BLADE-ClyA and J23101-ClyA culture overnight, at 37°C, in dark.
* Preparation for the next day:

+ Cast a 12.5 % SDS-PAGE gel (the following recipe is for 2 gels, and we used the BioRad Mini-Protean system).

- Mix the resolving gel (12.5 %) in the following order:

* 3.9 mL of MQ water
* 3 mL 1.5 M Tris-HCl, 0.192 M glycine, 0.1% SDS pH 8.3
* 5 mL acrylamide
* 120 µL 10% APS
* 24 µL TEMED
* (APS or TEMED must be added at last)

- Vortex the resolving gel solution, and pour 4.5 mL into the gel casting chamber. Work rapidly because the gel will polymerize quickly upon the addition of the polymerizing agents.
- Overlay the gel with isopropanol to get an event surface.
- After 45 minutes, the resolving gel is polymerized.
- Remove the isopropanol from the resolving gel.
- Mix the stacking gel (4 %) in the following order:

* 3.7 mL MQ water
* 1.5 mL 0.5 M Tris-HCl, 0.4 % SDS (pH 6.8)
* 0.8 mL 30 % acrylamide
* 60 µL 10% APS
* 7 µL TEMED

- Pour the stacking gel on top of the resolving gel, and insert the comb to make wells.
- After 30 minutes, the gel is polymerized and ready for use.

Second day:

+ Dilute the overnight cultures to 0.1-0.2 OD600, and grow the cultures until they reach 0.5-0.6 OD600.
+ Irradiate with blue light the appropriate samples for 4 h at 37°C.
+ Bacteria harvest:

- Pellet the bacteria at 4500 rpm for 30 min.
- Collect supernatant.

* Filter supernatant though through a 0.20-micron pore-size syringe filter.

+ Precipitate protein content with 60 % TCA.

- Add TCA at a final concentration of 10 %.
- Incubate with TCA overnight at -20°C.

Third Day:

+ Centrifugate protein at 4700 rpm for 30 min.
+ Remove supernatant and add 200 µL cold acetone to proteins, and Centrifugate protein at 14000 rpm for 5 min (2x).
+ Incubate at RT until the acetone evaporates.
+ Remove supernatant and resuspend proteins in 30 µL PBS.
+ SDS-PAGE analysis:

- 6x Laemli for 20 µL sample.
- Boil samples for 5 min at 100°C.
- Run the gel on 180 V, 45 mA for 40 min.
- Paint it for 20 min with Coumassie dye ( 0.24 % CBB-R250, 50 % ethanol 9 % acetic acid)
- Wash it overnight with Destaining solution (17.5 % ethanol, 12.5 % acetic acid)

---

# Blood agar hemolysis assay

First day:

+ Incubate J23101-BLADE-ClyA, J23101-ClyA, and J23101-BLADE-mCherry cultures overnight at 37°C in dark.

Second day:

+ Spread cultures on blood agar plates, and incubate them for 12 h in dark at 37°C.
+ Irradiate the light sample plates for 4 h at 25°C, while you kept the control plates in dark, at 25°C.

---

# mCherry production measurements

### Instrument I.

First day:

+ Incubate J23101-BLADE-mCherry culture overnight in LB medium at 37°C, 250 RPM, in black plastic tubes.

Second day:

+ Measure optical density at 600 nm (OD600), dilute it to 0.1-0.2, and allow it to grow for 2 h (until the OD reaches 0.5-0.6).
+ Complement media with 5 µM FAD (DMSO content must be 1% or below).
+ Start the induction with blue light.
+ Take 200 µL samples every half hour, and store them in a 96-well plate on the ice.
+ Measure mCherry (SpectraMax ID3 Multi-Mode Microplate Reader, Molecular Devices).

- excitation: 580 nm
- emission: 610 nm

### Instrument II.

First day:

+ Incubate J23101-BLADE-mCherry culture overnight in LB medium at 37°C, 250 RPM, in black plastic tubes.

Second day:

+ Measure optical density at 600 nm (OD600), dilute it to 0.1-0.2, and allow it to grow for 2 h (until the OD reaches 0.5-0.6).
+ Complement media with 5 µM FAD (DMSO content must be 1 % or below).
+ Plate cells to 96-well plate.
+ Start the induction with blue light.
+ Take 200 µL samples every half hour, and store them in a 96-well plate on the ice.
+ Measure mCherry (SpectraMax ID3 Multi-Mode Microplate Reader, Molecular Devices)

- excitation: 580 nm
- emission: 610 nm

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4375/wiki/titles/results-title-01-min.png)

Our project, **NanoBlade**, is an engineered *E. coli* capable of **detecting cancer cells** and eliminating them locally upon **blue light** irradiation. Accordingly, it comprises two elements: a detecting device called **Nanobody Display System** and a drug delivery device, the **BLADE Expression System**, which secretes toxins into the tumor environment upon blue-light induction.

When we designed NanoBlade, one of our main considerations was to make it highly modular because this way, we could evaluate the features of each system individually. (Fig. 1.)

![](https://static.igem.wiki/teams/4375/wiki/results/res-done-1.png)

During our work, we could characterize neither of our detection devices, as we could not assemble our Nanobody Display System. Fortunately, we could its successor, the **Mininano System**, but it did not arrive on time, therefore, we had no time left to study it in depth. (If you wonder what difficulties we have faced, please visit our  [Notebook page](https://2022.igem.wiki/elte/notebook).)

Although by overcoming great difficulties, we were much more successful with **BLADE Expression System**, which has allowed us to carry out many experiments. In the following, we will concentrate on these results.

# Assembly of the BLADE constructs

We ordered the sequences from iGEM sponsor **IDT**, and as they arrived, we digested their ends with XhoI and XbaI restriction endonucleases. This was done similarly to pETARA plasmid, which we chose as the plasmid for our constructs (Fig2/B, C). Ligation was done in a standard 3:1 insert:vector ratio with T4 ligase, then transformation followed into competent Xl1Blu cells. As colonies were visible, we isolated the plasmids and confirmed our results with diagnostic digestion, which gave us positive results (Fig2/D).

![](https://static.igem.wiki/teams/4375/wiki/results/res-done-2.png)

# Modeling blue-light inducible production with mCherry

With mCherry production, we aimed to study light-inducible protein expression. This protein seemed to be ideal for this purpose since it was also used for BLADE characterization and has a similar size to Cytolysin A (34 kDa), which we have planned to deliver into the tumor environment. [1, 2]

![](https://static.igem.wiki/teams/4375/wiki/results/res-done-3.png)

**Team ELTE has developed two types of illumination devices**, both capable of emitting blue light around 450 nm. (To know about them, please refer to our  [Hardware page](https://2022.igem.wiki/elte/hardware)) The first is a quite simple device with whom we could monitor the mCherry production over time at a given light intensity. Fig. 3/A shows the results of this experiment. After 7.5 hours of irradiation (at 37°C), we observed an 8.25 fold increase in fluorescence which was significantly less than was described in the literature. (15x) However, this could be easily explained by the difference between light sources since Romano et al. used an illumination setup that produces an irradiance of 5 W·m−2. [1] We have not obtained such information about our device yet, therefore, these results cannot be compared exactly.

Our other illumination device is capable of irradiating a 96-well plate and is highly programmable. With it, we could investigate the light intensity-dependent nature of our protein production. (Fig. 3/B) We could detect differences between illuminated and dark samples during this experiment, but significantly less than we previously did. This also could be reasoned by the different light sources or the different temperatures which we used for the protein expression. (37°C vs. 25°C) Moreover, we could not put our second device into an incubator, the culture was not as oxygenated as in the first case.

Later we hypothesized that the difference could be explained by the different bacterial culturing protocols, which could affect the number of plasmid copies. The previous measurement was conducted with a freshly transformed culture, but in the other case, we used frozen glycerol bacteria stock.

We observed a slight difference between protein productions at different light intensities; they leaned into saturation which met our expectations. It seemed, over 57% of power, that there was no substantial difference between samples.

# Cytolysin A production of the BLADE Expression System

![](https://static.igem.wiki/teams/4375/wiki/results/res-done-4.png)

We aimed to characterize the Cytolysin A production BLADE Expression System using SDS-PAGE, and study its hemolytic activity on blood agar plates. (Fig 4.)

For the Cytolysin production characterization, we obtained the supernatant of the bacterial culture after 4 h of blue light illumination and precipitated its protein content. We observed a band indicating Cytolysin A production, where we expected, which was absent in the case of the dark control sample. (Fig. 4/B) We implemented the protocol of Chiang et al. for this aim, but it should be noted, that this experiment needs to be repeated with a construct to which an affinity tag, such as 6xHis-Tag, is added. [3] In this way, the protein of our interest could be purified, lessening the number of aspecific bands.

We also meant to assess the hemolytic activity of Cytolysin A, using the BLADE Expression System. We spread the overnight cultures on Blood Agar plates, grew them for 8 h in dark at 37°C, which was followed by a 4 h long illumination with blue light at 25°C. (The dark control plate was kept in dark at 25°C for that time.) For additional negative control, we also spread BLADE-mCherry sample on the plates.) As we expected, no hemolytic activity was observed in the case of mCherry. Regarding the BLADE-ClyA samples, the biological activity of Cytolysin A was undoubtful since the blood agar discolored where the samples were plated. Unfortunately, we could not observe a significant difference between dark and illuminated plates. This could also be explained by the considerations we beforehand mentioned (Temperature, frozen bacterial culture).

# Preliminary experiment for ClyA cytotoxicity characterization on cancer cell culture

![](https://static.igem.wiki/teams/4375/wiki/results/red-done-5.png)

As part of our future experiments, we would like to study the cytotoxic effect of light-inducible ClyA production on cancer cells. For this aim, we did optimization measures of a widely used resazurin-based cell viability assay, where we assessed the optimal cell number (around 6000 cells/well) which should be plated on a 96-well plate for the experiment we would like to conduct in the future.

# Future Experiments

We appended Fig 6. to demonstrate the future of NanoBlade; from this point to market approval.

![](https://static.igem.wiki/teams/4375/wiki/results/res-done-6.png)

---

[1] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. *Nat. Chem. Biol.* **2021**, *17*, 817–827. <https://doi.org/10.1038/s41589-021-00787-6>.

[2]  [http://parts.igem.org/Part:BBa\_K811000](http://parts.igem.org/Part%3ABBa_K811000)

Chiang, C.-J.; Huang, P.-H. Metabolic Engineering of Probiotic Escherichia Coli for Cytolytic Therapy of Tumors. *Sci. Rep.* **2021**, *11*. <https://doi.org/10.1038/s41598-021-85372-6>.

{% endblock %}