Design

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.

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.

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.

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

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.

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

Participant: Fanni Englert

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

June 24 - 29

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.

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

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

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.

Participant: Fanni Englert

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

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

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

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.

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.

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

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).

No colonies were obtained.

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 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.

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

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.

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.

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

07.31. - Worldshaper-HZ

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

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

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.

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

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.

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.

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

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.

Participants: Laura Király, Zsóka Csorba

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

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.

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.

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

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

DNA work

Participants: Laura Dénes, Adrienn Bíró, Ágnes Golarits

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.

Participant: Fanni Englert

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

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.

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

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) and we ordered from IDT. Thank you, IDT!

Participant: Fanni Englert

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

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

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

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.

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

Participants:Laura Király, Zsóka Csorba

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

09.15. - MSP-Maastricht

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

September 19 - 23

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.

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

Participants:Zsóka Csorba, Laura Dénes

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

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

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.

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.

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.

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!