Difference between revisions of "Team:Vilnius-Lithuania/Human Practices"

Stage 1: Creating the team

In our team, we have clearly understood the importance of the whole team dynamics, work planning, and responsibilities allocation. The organizational architecture of our team's resources is based on strategic planning activities. To appropriately plan our project development timeline, we have consulted different stakeholders that contributed to our team functioning in one or another way. We have worked in close collaboration with the private sector – our general sponsor Thermo Fisher Scientific Baltics, and the public sector – our Alma mater Vilnius university.

From the very beginning, we have understood that the coordination of the whole team that, in our case encompassed of laboratory, human practices, IT, design, mathematical modeling, marketing, and public relations areas, requires a defined, coherent management system. Therefore, our team management and formation activities had two directions: formal and informal. The former was aimed at outlining precise team objectives, allocating responsibilities and workload; the latter was intended to promote various team building activities.

Team-building

We started our journey to Boston in mid-January when the new team of Vilnius-Lithuania iGEM gathered. The first team forming activity under informal direction umbrella took place in mid-February when the whole team had a workshop on team dynamics and conflict management. The workshop was led by a professional facilitator and consultant on strategy management Mrs. Eglė Daunienė, an Associate Professor of Vilnius University, and was mainly focused on the formation of the team at different stages of its development.

Everyone was asked to name 'my why' – the purpose of joining the team and expectations for the following year. This exercise is designed to make everyone feel the freedom to express their ideas and accept positive and negative feedback.

Another focus point of the workshop was team roles. Each team member was provided with the theoretical descriptions of four role areas: 1) interpersonal roles; 2) informational roles; 3) analytical roles; 4) acting roles.

Finally, we were told what problems may arise and what are the ordinary stages of a winning team. Team members were introduced to Tuckman's stages of group development: Forming, storming, norming, and performing.

Read more in our Team Building Handbook

Implementations:

• Understanding of Tuckman's model at the very beginning of our team forming stages helped a lot in the future. One of the most important aspects was realizing that storming is a normal and even crucial stage of successful team development. However, if solved right, each conflict leads to an improvement in working. It is essential to understand that the storming stage comes to each team, and the worst choice is to ignore it. In our group, we had a rather long storming phase, understanding principles of team development helped us not to avoid conflicts but form healthy team relationships from the early beginning.


• The mapping of our team interpersonal role matrix showed that we have all types of people in our team, which turns out to be the best possible composition of the team, aiming to achieve common goals.


• Therefore, Vilnius-Lithuania 2019 iGEM team was able to reach the peak of performing during autumn. We now know how to use everyone's potential, and this has lead to completing our project the best way we could.

Photos from team building workshop

Strategic plan preparation

One of our first meetings in iGEM was with members of our long-term sponsors – Thermo Fisher Scientific Baltics. During the session, we were encouraged to make a strategic plan for a year, as it would help us to better coordinate work and follow our progress.

After the meeting, we were introduced to Senior Business Excellence Manager Evelina Markauskė, who told us about the methods and philosophy implemented in Thermo Fisher Scientific. She also taught us about the X-Matrix, Lean Tools, Lean Management, and A3 Management.

Read more in our Strategic Plan Handbook

Implementations:

• After the meeting, our team prepared this year's plan.
• We have structurized our weekly meetings.
• We started filling a weekly plan and a progress table.

Photos from meeting with Senior Business Excellence Manager Evelina Markauskė

Forming Vilnius-Lithuania iGEM 2019 team

At the beginning of 2019, a group of students was selected by Vilnius-Lithuania 2018 iGEM team. This happens every year – the last year's team prepares a questionnaire and runs an interview session to choose new members from various disciplines, including math, computer science, politics, medicine, and, of course, biology and biochemistry. This is because every Vilnius-Lithuania iGEM team was, is, and will be entirely student-run and diverse, and we consider this our biggest strength. The PI, instructors, advisors, and anything else the team needs are selected during the year and depend on the project's main idea and the help needed.

Photos from interview session

Receiving good practices from previous Vilnius-Lithuania iGEM teams

The first month of the newly assembled students was dedicated mostly to meeting with the participants of the previous year's competition and learning from them as much as possible about iGEM in general, idea development, marketing strategies, PR, and other smaller, but crucial, things. Without these talks, we would not have been able to navigate during the first few months.

Implementations:

• The team decided to spend January gathering knowledge about synthetic biology and other iGEM projects, only then progressively move on to generating the idea. This meant studying from the edX course "Principles of Synthetic biology" and reading the most recent articles in the field of synthetic biology.
• During our weekly meetings, we presented the best iGEM projects to each other and discussed them to learn about the types of projects and what can be done in a year.
• The new team agreed to organize Biohackathon for the second time.
• We were recommended to do team-building with the help of professionals.
• Members with no previous experience got accustomed to the working discipline and tempo that a competitive iGEM team requires.

Photos from meetings with the participants of the previous year's competition

Shaping our idea by meeting with potential stakeholders

Our team spent the whole of February and half of March brainstorming to find the best idea for this year's project.

A plethora of very different ideas was proposed during this time:

1. An expression system to maximize secreted toxic protein yields


2. A reporter-based system for observing the protein of interest dynamics (expression level, degradation, aggregation, and toxicity effect on a cell) in real-time.


3. Biological polymers as a way of information storage


4. Adaptive laboratory evolution for optimization of strains synthesizing membrane proteins


5. Repressor-based light-inducible one-component systems


6. De novo enzymatic DNA synthesis


7. In silico metagenomic mining

To select the best idea and gather the opinions of specialists from different biology-related fields, we sought some of the best minds the faculty we are based in, the Life Sciences Centre can offer. We organized meetings with Prof. Saulius Serva, Dr. Paulius Lukas Tamošiūnas, Prof. Virginijus Šikšnys. Conversations with them helped us develop a better general understanding of their respective subjects and recognize the possible practical risks which they had faced and might be potential threats to our project.

We came to the conclusion that the best idea is the creation of light-inducible bacterial systems using in silico metagenomic mining.

After coming up with the primary concept, we wanted to discuss it with the people whom this project may impact:

• The scientific community


• Companies


• The general public

Prof. Rolandas Meškys

Because we wanted to work with metagenomics and create systems that would help in various areas of biology, including biocatalysis, our team reached out to Professor Rolandas Meškys, the head of the Department of Molecular Microbiology and Biotechnology in Life Sciences Center.

Prof. Meškys encouraged us to follow through with this idea as it is a growing field with huge potential. He asked about the applications of optogenetics in bacteria, and while discussing it, we came up with a way to apply light-inducible bacterial systems in cancer therapy. We postulated that this would allow treatment to be surgically non-invasive by allowing highly precise therapeutic protein release inside the tumor.

The professor also suggested not to limit ourselves with the search of new possible tools, but also, by applying rational design, develop optogenetic tools from known proteins.

Since Prof. Meškys had such great insight, we asked him to take the role of our Principal Investigator, and he gladly accepted. Vilnius-Lithuania 2019 iGEM team feels very proud to have such an established member of the academia as our PI.

Implementations:

• Together we came up with the Idea of application - light inducible bacterial systems in cancer therapy.
• Decided not only rely on in silico metagenomic mining but try rational design for creating novel optogenetic tools.
• We welcomed our new PI.

Photos from meeting with Prof. Rolandas Meškys

Dr. Darius Kazlauskas

We had a meeting with Dr. Darius Kazlauskas, who is a scientist working with in silico metagenomics. In one of his projects, he tried to assemble viral genomes from various metagenomic datasets. He worked with Ebi-metagenomics, IMG/M, iMicrobe.us. However, he had problems with the biggest metagenomic database MG-RAST, which stores more than ten times more information than all the other databases together.

According to him, there are massive problems with some databases, especially MG-RAST, as the data is not structured. Moreover, data storage and analysis requires enormous amounts of system resources and advanced expertise in the field. Therefore, we decided to work further with the MG-RAST database for gene mining of novel optogenetic tools. We made an aim to open up this metagenomic database for scientists with no prior IT knowledge and to provide a means to develop new parts in synthetic biology.

Implementations:

• Decided to work with MG-RAST

Photos from meeting with Dr. Darius Kazlauskas

Thermo Fisher Scientific Baltic

It is essential to gather as many different opinions as possible, and what can be more different than business and academia. Our team wanted to find out about the potential applications and marketability of our project from the business point of view. So we organized a meeting with Dr. Remigijus Skirgaila, a scientist from Thermo Fisher Scientific, and his colleague Dainius Tautvaišas, an iGEM judge, who helped to organize this meeting and often provided our team with general consulting.

Team members were pleased to hear that optogenetics might be the key to make the regulation of bacteria easier. Dr. Skirgaila saw our project as being technically implementable, as well. We also learned that as far as they know, bacterial control is only possible at the transcriptional level.

Moreover, they were very interested in our metagenomic sequences mining software. They told us that we should seek the development of this software thoroughly as it would open up a possibility to access a considerable amount of data. They also suggested to do a cloud-based system and to contact with cloud offering companies such as Google.

Implementations after a meeting:

• We understood that in silico metagenomic mining has considerable potential.


• Our team contacted Google to receive a cloud for performing computations.


• We started to discuss the possibility of control not only at the transcriptional but also at the translational level.


• The team started to think of ways to make our project more marketable.

Photo from meeting with Dr. Remigijus Skirgaila from Thermo Fisher Scientific Baltic

Lithuanian School Student Union

Vilnius-Lithuania 2019 iGEM team had a chance to participate in the Lithuanian School Student Union (LMS) organized event – Student Organization Fair in Memory of the 30th Anniversary of The Baltic Way.

I fear that even if I were able to create the most nonsense idea for my book, scientists would find a way to say – well, that is already possible!

In the event, we created a quiz inspired by famous Lithuanian public figures - publisher Tomas Ramanauskas and philosopher Kristupas Sabolius who run an online channel to foster pupil creativity. They took synthetic biology as an example of a source of inspiration for writing a science fiction novel. One of their concerns took our attention. “I fear that even if I were able to create the most nonsense idea for my book, ” T. Ramanauskas explained, “scientists would find a way to say – well, that is already possible!”. Inspired by this idea, we wanted to see whether people could distinguish real synthetic biology projects from futuristic ideas and science fiction, while also having a meaningful conversation with them. This way, we were aiming to introduce the possibilities of science through some examples of applications.

During the event, we gave participants six synthetic biology projects, including a description of our iGEM project. Their task was to select two projects that have already been completed, two which are currently being developed, and two ideas that fall under the science fiction or futurism tab.

For the already completed projects, we introduced digital information coding in bacteria genome and synthesis of spider’s silk in yeast.

We talked about how information coding in the bacterial genome will prevail in the future since we will be short of data storage resources soon. Another presented project was spider’s silk that is produced by yeast – researches have created new material that mimics spider silk’s strength, stretchiness, and energy-absorbing capacity. Surprisingly, the majority of participants had already heard of these inventions and were able to distinguish them quickly.

The second part included projects that are being developed. We described our iGEM project (light-controlled bacteria). Interestingly, some people described it as futuristic, as we described it more as a fundamental project and didn’t mention application fields. The project that captured the participant's attention the most was bioluminescent plants and algae. Explaining the main steps of gene cloning and giving some examples of inserting fluorescent protein-coding genes to plant genome, we were seeking to introduce people to applications of synthetic biology in everyday life. Plants that could work as night lamps brought immense attention not only from young people but also from the elders, who thought that would be suitable for home decoration.

To represent futuristic projects, we chose the Designer Baby project, which would enable parents to choose their desired characteristics for their future children. Most of the participants immediately distinguished it as not yet possible. However, when discussing the possibility of altering a baby's genetic make-up, people found themselves in a moral quandary. Some would like to use this application to avoid hereditary illnesses. Still, the majority of people saw it as an unethical and unsafe procedure. The last project we presented was about synthetic cells that could be designed and programmed over a personal computer and then created from scratch. Most of the time, participants choose this project as already developed. People mostly thought it was the same as genetically modified organisms. Although some of them were not familiar with the method of GM organism's creation and after our explanation, they were enthusiastic to know more.

Implementations:

• We noticed that people happen to distinguish synthetic biology projects as futuristic when everyday life application is not provided. Therefore, in our future presentations, we included more examples of synthetic biology's practical use.
• Even though participants knew some facts of biotechnology and synthetic biology, the majority still lacked some background knowledge about biotechnology methods and principles of biology. Thus, we decided to include more practical activities in our future events.

Photos from Lithuanian School Student Union (LMS) organized event – Student Organization Fair

Student for one day

Vilnius-Lithuania 2019 iGEM team participated in the Vilnius University event Student for One Day. During this event, we not only presented our project and synthetic biology in general but also included some practical tasks.

High school students were given a chance to do some laboratory work and at the same time, learn about the methods and principles of biotechnology. They performed an experiment to incorporate urease into alginate capsules and then used these capsules to detect urea. This way, they learned that urease hydrolyzed urea to ammonia and carbon dioxide and were able to gain an understanding of indirect measuring methods (in this case, change in pH to detect urea). This was applied to estimate water pollution in various samples.

After the practical tasks, the students split into groups to participate in a synthetic biology quiz, which further deepened their understanding of possible biotechnology applications. Their final exercise was to think of a real-world implementation of our light controlled bacterial system (as our team members were explaining the mechanism of the system before, they skipped this part on purpose). Real iGEM participants blended with the student teams to act out a brainstorming and idea pitching session. And even though students regarded the task as challenging, they were able to create genuinely original ideas. The most imaginative idea was about a bacterial strain that could be used for repairing buildings. As light penetrated slits in a ruptured brick, it would activate a response in bacteria for secreting appropriate substances, thus fixing the break.

Implementations:

• Simple, practical exercises attracted the pupil’s attention when explaining biotechnology methods and presenting synthetic biology. Therefore, we included more practical experiments to explain theoretical principles in our future presentations.

Photos from the event Student for one day

International Conference The COINS 2019

Both 2018 and 2019, Vilnius-Lithuania iGEM teams participated in an international conference of life sciences The COINS. Here we attended company fair and made both oral and poster presentations of the 2018 Vilnius-Lithuania iGEM team project.

More than 300 students attended the presentation to hear about iGEM competition and synthetic biology. This presentation was the very first from Vilnius-Lithuania iGEM 2018 to our local community after they had come back from Boston.

The attention of participants was captured not only by the last year's but also by the current team. New iGEM participants were reaching out to potential sponsors, mentors, communicating with interested members of the academia.

Most importantly, our team had an exclusive opportunity to talk with the Nobel prize laureate in Physiology or Medicine in 2014, Prof. John O'Keefe, and hear the story of his life, research, as well as the path of becoming such an esteemed member of the scientific community. He talked passionately about the importance of interdisciplinary thinking, especially philosophy, and not giving up when it seems rough.

Because Prof. O'Keefe is a neuroscientist with a background in engineering, he was able to point out the benefits of implementing engineering principles while working with biological systems. This was important to us, as we were already thinking of working with optogenetics, the start of which is also closely related to neuroscience.

Also, we took the professor's advice on interdisciplinarity, therefore later on we tried to discuss our project with mathematicians, physicists. Moreover, every time organizing an event, we tried to invite as intellectually diverse people as possible (for example, we asked the philosopher Dr. Jonas Čiurlionis, to participate in our discussion on the future of humanity and technology).

Photos from International Conference The COINS 2019

International Biology Olympiad

Vilnius-Lithuania 2019 iGEM team did not miss a chance to help students preparing for the International Biology Olympiad. We organized a practical work activity based on Practical Paper 2 from the 2015 International Biology Olympiad.

During the practice, students performed standard lab procedures, such as PCR, restriction analysis, and gel electrophoresis. The experimental work required critical thinking and previous knowledge as the participants had to choose the required temperature, restriction enzymes, and primers. When they finally got the results, they learned how to interpret them and make the right conclusions.

After the experiments, we examined the main mistakes. Then we showed tips and tricks preparing reaction mixtures for PCR and restriction analysis and together discussed the results. Afterward, the students were introduced to iGEM competition, synthetic biology, explained to why and how the principles of engineering and Boolean logic are used in the process of creating a genetically modified organism. Moreover, we invited them to join the competition during their studies at Vilnius University!

Photos from International Biology Olympiad

Building Colight

We saw a massive gap in the optogenetics field as most of the transcriptional regulators by light are two-component systems, which have a considerable disadvantage as they require a vast amount of cellular resources, are slowly modulated, and have a poor portability.

Currently existing one-component light modular systems have a variety of serious drawbacks, such as low dynamic range, slow response time, lack of portability, and a lack of parts. What is more, it is only possible to modulate bacteria dynamics at the transcriptional level.

We also saw a problem in in silico metagenomic mining, as the sequences we wanted to analyse seemed to require expertise in the field. Most of the tools we used at first had a difficult or not user-friendly interface and a lot of input and output data needed to be processed manually.

The described problems became the basis of the following tasks of our project.

Kotryna Kvederavičiūtė

In March, the team had a meeting with Ph.D. candidate Kotryna Kvederavičiūtė who works in the field of bioinformatics to discuss our approach to in silico metagenomic mining.

Firstly, Kotryna suggested finding access to enough computer memory and computational power to store all the necessary data and run the algorithms needed for our software. Together we found a way how to solve this problem. Our team members asked Prof. Juozas Rimantas Lazutka if we could link together the computers in the Life Science Center computer class to perform our calculations.

We also settled on using Blast or HMMER algorithms for our software and that the best way would be to search among already translated sequences.

We discussed the possibilities of using different platforms for searching sequences with protein domain profiles.

Implementation:

• We settled on using Blast or HMMER algorithms for our software and that the best way would be to search among already translated sequences.

Photos from meeting with Kotryna Kvederavičiūtė

Prof. E. Sužiedėlienė and Dr. Justas Dapkūnas

Having the idea that we could extract light-modular transcription regulators from metagenomes, we searched for the way how to find sequences which our newly discovered protein bind to.

Our team met Dr. Justas Dapkūnas, who works in the field of bioinformatics as a researcher in the analysis of protein structure, surface properties, and interactions. We reasoned that by using bioinformatic tools, we could predict a DNA sequence that a protein of interest could bind to.

However, team members heard from Dr. Dapkūnas that this sort of protein structure modeling is a possible but extremely challenging task. The first difficulty stems from the fact that sometimes completely unrelated protein bind to the same DNA sequences, and other times homologous protein bind to entirely different DNA sequences. Secondly, it could be possible to predict the DNA sequence by docking a very closely related protein with a known structure to genomic DNA from the organism the protein of interest comes from. However, this method would not be possible, as our protein sequences would be gathered from metagenomic databases.

After this conversation with Dr. Dapkūnas, we understood that we would not be able to find the needed DNA sequence in silico and would have to find them by performing in vitro experimentation. Therefore, we asked Prof. Edita Sužiedėlienė to give us a piece of advice. She suggested performing double-stranded SELEX (Systematic evolution of ligands by exponential enrichment). This approach started by expressing our protein of interest fused with an affinity tag in E. coli. The next step would be immobilization of the protein on beads and bringing double-stranded chemically synthesized random nucleotide containing oligonucleotides. Professor Sužiedėlienė warned that the assay might be noisy; therefore, we should be prepared to optimize the conditions as much as possible.

Implementations:

• We understood that finding DNA binding sites in silico would be too challenging. Therefore, we decided to use in vitro method.
• We decided to use the double-stranded SELEX method to find the protein binding sites of the undescribed protein of our research.

Photos from meeting with Prof. E. Sužiedėlienė and Dr. Justas Dapkūnas

Irmantas Rokaitis

We decided that the best approach for searching for the protein binding sequence in DNA would be running five cycles of SELEX followed by EMSA (Electrophoretic Mobility Shift Assay) to separate the oligonucleotides that attach to the protein and those that do not. However, standard EMSA requires a radioactively labeled DNA sequence.

Irmantas Rokaitis is an MSc Student of biochemistry, currently working with the EMSA technique. As he has been a member of two previous iGEM teams, we decided to invite Irmantas to discuss lab safety. He told us that ordering radioactively labeled nucleotides is only possible under specific circumstances. He also said that if we were able to get everything needed, we should find a lab space that is suitable for this sort of work and meets all the requirements.

As an alternative to radioactively labeled nucleotides, he suggested us to use fluorescent-labeled DNA. Even though it has a lower sensitivity, it would be a much safer alternative.

Implementations:

• We settled on using fluorescent-labeled DNA for our following experiments instead of radioactively labeled nucleotides.

Photos from meeting with Irmantas Rokaitis

Modeling

Early on in the modeling process, our team members met Prof. Olga Štikonienė and Prof. Artūras Štikonas. They directed us towards useful literature in the theory of nonlinear dynamical systems, delay and stochastic differential equations, control theory, and their applications in modeling of biological processes. Their help was critical, as it opened the way to use these tools in our work.

We asked a roboticist, Paulius Briedis, to suggest the best way to estimate the system state from known input-output relationships. According to him, for single-input-single-output systems, we could use nonlinear regression, whereas for more complex situations, a machine-learning approach, such as a genetic algorithm, was more viable.

Difficulties in making sure that our photokinetic reactions were accurate and our inability to get correct results in computer simulations prompted us to meet Prof. Dr. Saulius Bagdonas, who works at Vilnius University Laser Research Center. The professor quickly identified the source of our confusion. He explained that various researchers express quantities in different dimensions, so we should dimensionally renormalize the equations. We have also learned how to calculate (approximate integrals of) effective light intensities, the most decisive factor in photochemical reactions, and that our modeling approach to photokinetics was mostly correct.

Implementations:

• The theory of nonlinear dynamical systems and control theory were used extensively throughout the modeling process.
• We started using dimensional analysis to understand photokinetic equations.

Biohackathon

During our annual Biohackathon: Lab issues event, four members of the Vilnius-Lithuania iGEM 2019 team were participating as an individual team; however, they could not compete for any of the prizes. This allowed us to get numerous consultations and discussions with top-level scientists and engineers in the field.

The team's initial software idea was to build a metagenomic analysis desktop app for finding a protein with desired characteristics that would use local system resources and would work on any operating system. Before even starting to write the code, our team had a set of concerns about the software being a desktop app, data cleaning, and hardware problems like storage and processing power.

Photos from BioHackathon

Michail Chrunov

After having our first consultation with Michail Chrunov, desktop software developer, a decision was made to create the software as a web service application. According to Mr. Chrunov, this would increase the reachability and would be more convenient for those who just want to try it without having to install or download additional files. Additionally, this way, the app would use our hardware so we could be sure the analysis to be completed without any hardware-related errors.

Implementation:

• We decided to build software as a web-service application instead of a desktop app.

Photos from BioHackathon

Dr. Justas Dapkūnas, Dr. Kliment Olechnovič, Kotryna Kvederavičiūtė

The next major challenge was solving one of the biggest problems in metagenomics, that is, removing the duplicates from the multi-million sequence FASTA format files and to standardize the sequences for further analysis. Although at first glance, it seemed like a simple enough problem, we later found out that it is a well-known ongoing issue within the bioinformatics field. We had lengthy consultations and discussions with Dr. Justas Dapkūnas, Dr. Kliment Olechnovič, and Kotryna Kvederavičiūtė, and it took us numerous hours until we came up with an algorithmic solution for the problem. After implementing it and improving the code, we ended up having a tool that not only removes duplicates but also standardizes and cleans up the data, which is a must-have for a flawless analysis.

Implementation:

• Solved the duplicate problem and implemented standardization & cleanup algorithms.

We then moved on to tackling the problem with space for data storage. Since metagenomic data collections take up terabytes of data, we figured that it wouldn’t be cost-effective to let every user store their data on our servers. Dr. Dapkūnas suggested that we could use the best-in-class clustering tool for this job, which takes out only a certain percentage of representative sequences and clusters them into a new, up to 70% smaller file. We were also told that if we are working with big data, the Apache Spark cluster-computing framework is a must.

The general discussions about our software included topics such as application, performance, legal rights, value, and its impact on the scientific community and the need for such software. We took in many opinions and suggestions from real professionals of their fields and came out of this event with a result we are proud of and hope it can make a breakthrough in the scientific community.

Photos from BioHackathon

Dr. Nicholas Ting Xun Ong

Our laboratory experiments showed that Gal4-QPas1 and LexA-QPas1 chimeras repress promoters up to 3 times, which did not quite meet our expectations. To improve our system, we decided to seek advice from a scientist with expertise in working with BphP1. Therefore we found Dr. Nicholas Ting Xun Ong, whose Ph.D. study was mainly focused BphP1/PpsR2 optogenetic system in E. coli.

The response that we got was that, in general, trying to build chimeric proteins to function as transcriptional regulators, e.g., an engineered PpsR2 repressor, is not a very easy task. Dr. Ong also suggested taking a look at his former lab's recent publication about such efforts.

Dr. Ong's research shows that the problem with the low dynamic range between the induced and the uninduced states is not linked with PpsR2 binding to DNA. Instead, the key factor is how BphP1 interacts with PpsR2, that is, how BphP1 disrupts the dimeric structure of PpsR2 in the light-excited state. In one of his papers, he was able to show 76 fold repression by using PpsR2; however, BphP1, even after a thorough optimization, derepressed only 2.5 fold.

Dr. Ong recommended trying a high expression level pBR_crtE promoter and was very optimistic about our idea to try Q-Pas1, a minimal PpsR2 domain, fused with a DNA binding domain. He said he has considered this idea, but due to a lack of time did not try it.

Implementations after the consultation:

• We decided to try Q-Pas1 fused with the HTH domain from the PpsR2 protein.


• To work with the pBR_crtE promoter, offered by Dr. N. Ong.


• Knowing that all the promoters Dr. Ong used had two operator sequences, we decided to test new promoters, designed based on the ColE promoter, with only one operator sequence.

Final Project

According to the feedback our team managed to implement our project from a transcriptional light-inducible system to a framework for a multilevel light-control in bacteria.

“Colight” - a multi-level collection of optogenetic tools for modular bacteria control. Our newly developed systems fulfill the gaps in optogenetic bacteria control in transcription regulation and open up a possibility of light control expression dynamics in plasmid copy number and post-translational levels.

Public

Vilnius-Lithuania 2019 iGEM team sought to communicate with the public as much as possible. We saw that shaping the public's understanding of synthetic biology is crucial for the future of our country and humanity. We sought to educate everyone from a pupil to a senior by participating in different events and tried to improve ourselves by receiving their opinion about our project.