Team:Brno Czech Republic/Poster

Presented by Team Brno_Czech_Republic 2020 from Masaryk University and Brno University of Technology, Brno, Czech Republic

Tomáš Kotačka¹, Barbora Hrnčířová ¹, Stanislav Juračka ¹,Petr Kohout ¹, Mgr. Karel Říha, Ph.D.², Mgr. Pavel Dvořák, Ph.D.², Ing. RNDr. Martin Marek, Ph.D.²

¹iGEM Student Team Member, ²iGEM Team Principal Investigators

Abstract: The goal of our project is to design a mobile device CYANOTRAP that degrades and detoxifies cyanobacterial blooms in lakes and water reservoirs. The core of the device are immobilized Bacillus subtilis that on their surface display enzymes required for the lysis of cyanobacteria cells and for degradation of cyanotoxins.

Such a design has several advantages over other solutions: 1) High mobility of the device would allow the targeting of the most infested areas, 2) Number of devices put in operation can easily be scaled according to the acreage of the water surface and degree of infestation, 3) Enzymatic lysis is more environmental friendly than chemical treatments, 4) The device can be used for constant surveillance and prevent formation of water blooms, 5) Use of bacteria as carriers of the enzymes provides longer sustainability as living bacteria can continuously replenish the enzymes.
Project Goals:

To design our device CYANOTRAP, that degrades and detoxifies cyanobacterial blooms in lakes and water reservoirs

To create a model of our device for basic analysis, which will allows us to peek inside the machine and observe what happens behind an opaque wall

To communicate with people about GMOs and science, even in entertainment way. Getting theoretical knowledge and practical experience is great, but the opportunity to share it is absolutely fantastic

To create a Handbook: How to handle Bacillus subtilis for other iGEM teams, and also improve the iGEM Team seeker
Due to the changes in the environment associated with human intervention, cyanobacterial overpopulation is affecting people and ecosystems all over the world.

There are two main contributing factors. Increased levels of phosphorus are a major factor as it is an essential substance, which cyanobacteria need to gain form the environment. Second factor is temperature as cyanobacteria multiply faster at temperatures over 25 ℃.

So what issues does cyanobacterial overpopulation cause?

  • Overpopulated cyanobacteria create so-called water blooms on the water surface, which can cause many issues for local animals.

  • The odor associated with the decomposition of the accumulated biomass.

  • Cyanotoxins, their secondary metabolites, are however the main issue. They are not only harmful to aquatic life, but also to mammals.

Our idea is to generate a small self-operating floating system called CYANOTRAP that is powered by solar energy and continuously lyses cyanobacteria and detoxifies water from their toxins. The core component of the device are engineered Bacillus subtilis cells that on their surface display enzymes required for the lysis of cyanobacteria cells and for degradation of cyanotoxins.

We will immobilize genetically modified bacteria on the surface of cellulose-coated beads that will be trapped in a chamber with filters. This will allow influx and efflux of water while preventing release of the immobilized bacteria.

Strain L produces a cellulosome, containing Lysozyme and microvirin. Its function is to lyse cyanobacterial cells. Strain D produces a cellulosome which contains Mlr enzymes to degrade the toxin microcystin. All enzymes are connected to the scaffoldin via dockerins and cohesins.
Basic parts: We first needed to determine the desired function of our device. Then we had to find out which DNA sequences would be able to perform the functions we had in mind.

Composite parts: After completing the list of our Basic Parts, we had to combine them into functional units.

Due to the SARS-CoV-2 pandemic, we only had time to focus on the Immobilization module (IM).The purpose of the IM is to immobilize the cells of B. subtilis on a cellulose matrix. This construct was designed to ensure that the genetically modified bacteria will not escape our floating device and endanger the environment.

Wet lab
Despite the adverse conditions in our country due to the coronavirus situation, we were able to demonstrate the functionality of some aspects of our CYANOTRAP project. Using tools of synthetic biology, we proved transfer of one of our synthetic constructs - firstly into the cloning strain of Escherichia coli and then also into the final host - Bacillus subtilis.

Scheme of our experimental workflow:

For the verification we performed agarose gel DNA electrophoresis of amplikons of the integration region of B. subtilis chromosome. Line 1 contains the IM. The lines 2, 3 and 4 contain PCR amplicons from the chromosomes of transformants of Bacillus subtilis (indicated by the blue arrow). Line number 5 contains negative control.

Proof by sequencing:
The presence of our synthetic sequence in the pDG3661 vector was also demonstrated by sequencing. We used the commercial service of the company SEQme based in the Czech Republic.

We also verified the integration of IM gene trough AmyE test, because pDG3661 vector integrates into the amyE locus and prevents transformed colonies from hydrolysing starch
Why do we want to model our device?
Modeling our device is cheaper, faster, and more effective than creating the physical machine. It allows us to peek inside the machine and observe what happens behind an opaque wall. It will also allow us to predict the possible complications.

We succeeded in creating a model of the machine where we can run a basic analysis like the collision between microbeads and cyanobacteria.

Future plans:
There are many ways to improve our simulation in the future.

  • To improve our model to better represent reality, we could achieve that by implementing fluid dynamics into our system and its impact on particular objects

  • To adapt our model, so it could answer a wider spectrum of questions (For example ‘Which volume of water we are able to proceed in an hour?)

  • To make our model very parametrizable to enable quick and iterable evaluation of settings for optimization of the design

As a contribution to iGEM, we've created a Handbook: How to handle Bacillus subtilis and iGEM Team seeker

We decided to write this easy and to-the-point manual for everyone who is working with Bacillus subtilis.

iGEM Team Seeker:
We decided to improve iGEM Team Seeker. Create a simple Team Seeker, which will include all teams that have competed so far, but will also be easy to update and expand its database to include future years of the competition.
  • We designed seven composite parts for B. subtilis.

  • We successfully assembled necessary synthetic construct carrying the immobilisation module.

  • B. subtilis was successfully transformed and the integration of our expression cassette into a specific region of its chromosome was confirmed.

  • We created a model of our device for basic analysis.

  • We contributed to iGEM by created Handbook: How to handle Bacillus subtilis for future iGEM teams and also with improved iGEM Team Seeker.

  • We also created a lot of educational materials and activities.


Future work
  • From the very beginning of our iGEM journey, we planned for CYANOTRAP to be a real product, useful in real life. We plan for CYANOTRAP to be a device floating on the water surface. The device itself will be controlled remotely by drone.

  • Next year, we plan to assemble the cellulosomes, transform Bacillus subtilis with the rest of our genes, and thus enabling our bacteria to display the scaffoldins on its surface.

  • To make CYANOTRAP even more efficient, we want it to accumulate phosphorus from the water by using engineered bacterial microcompartments (BMC).

  • There is also idea for a quorum sensing-based autolysis system, which would prevent genetically modified Bacillus subtilis from escaping into the environment.

Education and popularization of science is interwoven through our work. For us, the popularization of science is more of a hobby than a strenuous task. Also for this purpose, we created association Generation Mendel.

We are everywhere!
Facebook: #ScienceWednesday - weekly posts which include various themes related to our project, GMOs or synthetic biology
#ProjectFriday - weekly posts which are written in a bit more of a scientific language

Instagram: We used Instagram for posting informative pictures and graphics.

Twitter: There we shared some of our achievements and wrote about relevant topics.

Our Czech Website:
We used this website to provide information about us, about our project and also our educational materials and activities
Online curses: pop-science articles, covering a variety of biological topics
Magazine and Scientific coloring book: during nation-wide lock-down we created a short, biology-themed magazine with crosswords, word search puzzles and quizzes

← Retirement homes and children's homes which contacted us and used our Magazine or Scientific coloring book

Some of our educational events:
Lecture # IT'S not rocket SCIENCE: we held four lectures about GMOs

Children's Day
Acknowledgements and Sponsors

Prof. RNDr. Jiří Doškař, CSc.
Prof. RNDr. Zbyněk Prokop, Ph.D.
Prof. RNDr. Jan Šmarda, CSc.
Mgr. Tibor Botka, Ph.D.
Mgr. David Kovář, Ph.D.
Mgr. Libor Krásný, Ph.D.
Mgr. Geriš Rodan
Ondřej Beňuš
Bc. Gabriela Chmelařová
Bc. Veronika Kolajová
Vojtěch Lukáš
Jakub Svoboda

Mgr. Barbora Břenková
Mgr. Iva Buriánková, Ph.D.
Mgr. Barbora Burýšková
Mgr. Jana Faturová
Mgr. Adéla Indráková
Mgr. Marie Komárková
Bc. Jozef Kováč
Mgr. Anna Molíková