iGEM 2021 — Team TU_Darmstadt

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# Team:TU Darmstadt/notebook

Notebook – TUDA iGEM 2021

![](/wiki/images/8/89/T--TU_Darmstadt--Header_Export.svg)

Primary Menu

* [Home](./)
* Project
  + [Description](./description)
  + [Biofilm](./biofilm)
  + [Pathogen Sensing](./sensing)
  + [Bacteriophages](./phages)
  + [Biosafety](./biosafety)
  + [Contribution](./contribution)
  + [Engineering](./engineering)
  + [Proof of concept](./proof_of_concept)
* Team
  + [Team](./team)
  + [Collaborations](./collaborations)
  + [Attributions](./attributions)
  + [Sponsoring](./sponsoring)
* Human Practices
  + [Overview](./overview)
  + [Integrated](./human_practices)
  + [Education & Communication](./communication)
  + [Implementation](./implementation)
  + [Ethics & Policies](./ethics_policies)
* Modeling
  + [Genetic Circuit](./model)
  + [Phoenix](./phoenix)
  + [GoGEM](./software)
* Lab
  + [Results Summary](./results)
  + [Parts](./parts)
  + [Backbones](./backbones)
  + [Experiments](./experiments)
  + [Notebook](./notebook)
* Achievements
  + [Awards](./awards)
  + [Medal Criteria](./medal-criteria)
  + [Inclusion](./inclusivity)
  + [Safety & Security](./safety)
  + [Excellence](./excellence)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3985)
* [Plain Language](./plain-language)

# Notebook

## February

Week 6

Our team’s journey **started on the 6th of February** with our first meeting. We were all enthusiastic about the awesome iGEM experience we would share in this group and eager to find an awesome topic for this iGEM year.

This was officially our first week as the new iGEM Team of TU Darmstadt and we were extremely motivated to get started.

Prof. Jürgens from our university kindly offered to give us a talk about **how to read and write scientific research papers**. We were able to prepare well for the research ahead of us to find a project topic!

We started our research with a **meeting on the 13th of February**. We met on teams and went through a lot of SynBio papers to search for inspirations and ideas for our project. We **collected a lot of amazing project ideas**, such as an microbial alternative to wood, GMOs to prevent mite infections in honey bees, symbiotic rhizobacteriacea for improved crop growth and many more! We continued our research within the following weeks and met with our whole team each Saturday.

Week 7

We used the week to further **elaborate our findings from the first research weekend**. We met in small groups on teams, had great discussions and found a lot more information about our project ideas that could be presented on the next research weekend for the whole team. We still had **a lot to organize in our freshly founded team** and talked a lot about our general team structures and what we would do in the following iGEM year.

Week 8

This week we were again really busy with our **research phase**. Besides some general organization we mostly focused on scanning through research papers to elaborate our project ideas.

## March

Week 9

This week we were working hard on our **research phase** again. Also, we had a lot of discussions about our **team rules**: Since we are a rather big iGEM Team we had to ensure that we can stay productive and function well as one unit. Therefore, we created a set of rules everyone in the team committed to, to **work together respectfully in a fun atmosphere**.

Also, we were getting closer and closer to the decision of our project, so we all were pretty excited.

Week 10

After our intense research phase we were able to have a **discussion and vote between 6 amazing and well defined ideas**:

1. **Bioconcrete** as a biological engineered living material
2. Bacteria for electrical pulse induced **biosensors for HEV**
3. **Bioleaching** of rare elements
4. **Pathogen eradication** using phages
5. **TU Bee**
6. Inoculating **seeds with *Rhizobacteria***

We decided to continue our project with the topic **Pathogen eradication using phages**, tackling local and global **problems of MDR bacteria**, especially in extremely resistant biofilms. We were stoked to elaborate our amazing topic and **really get going with iGEM 2021!**

We started to work on our **project sponsoring** directly after deciding on a project topic on the 8th of march. We started by **collecting awesome companies** that might be willing to sponsor our iGEM Team and writing first drafts of contact forms.

Week 11

This week we had the **kick off events for both our Modeling and HP work**. We started by introducing both topics in our team meeting and continued with setting goals in these areas for the team.

We started our Human Practices work with an **introduction of Human Practices and its importance for modern research**. We were happy to contribute to a better understanding of synthetic biology in our society and to find applications of our project in the real world.

We started our Modeling team on the 15th of March on our team meeting. We were eager to contribute to our project with interesting ***in-silico* experiments**. Our first checkpoint was to get to know **programming languages** and **bioinformatic environments** such as Python or Rosetta.

Week 12

We were still pretty busy with **researching our topic** and getting more ideas for our iGEM project. We were also organizing our **small groups** for the research and project work ahead of us.

We were **introduced into Python** by Fran and Tom, members of 2019’s iGEM Team at TU Darmstadt. They are both experts in computational biology and were able to provide us with a lot of useful tips and tricks, for example regarding the **biotite bioinformatics package** for Python.

## April

Week 13

This week we scheduled the **kick off events for our research/lab groups**. We were all motivated to get experts in our topics.

To illustrate our wiki and presentations we also started with an **Inkscape workshop** with some of the members from last years team.

We started talking about the **iGEM medal criteria** and general **judging in the iGEM competition**.

Also, Eileen came up with the idea to have a **team plant**, that we can watch grow together just as our project grows. We choose **chard** as our plant, since its easy to handle and could be used in a **team cooking event** to cook a delicious recipe.

Week 14

This week the **research groups phage engineering, biosafety, detection and ECM degradation and killing** presented their research topics in our weekly team meeting. We were able to collect a lot of good points and to **discuss the problems or bottlenecks** that could come with this topics.

We also finished our **sponsoring portfolio**, introducing our team and our topic to companies. We wanted to contact companies as soon as possible to get support for our project both outside and inside the lab.

To get ready to plan our part assembly we **introduced SnapGene** to the team and scheduled a **SnapGene workshop** held by one of our project coordinators.

To push our research phase and coordinate our progress in the research groups with the whole team we had our first **project development meeting**. We used these Saturday meetings to have a presentation by every research group and discuss their progress with the whole team. This way everyone in our team had a **good overview over the whole project** and we ensured the research would lead into the same direction.

Week 15

Besides the **research in our research groups**, we collected ideas how our **Modeling team could support the project**. We therefore scheduled a **meeting with some iGEMers** of the previous years at TU Darmstadt and discussed our ideas. We got a lot of **inspirational feedback** from recent research papers and their experience.

Since we got **no lab access at least till July** we **discussed the idea** from one of the ambassadors at the iGEM ambassador meeting to have a **two phase project** with two phases: Design and Model in the first year followed by Build and Test in the second year.

Week 16

We got into contact with a lot of **experts in the fields of phages, biofilms, biosafety e.g.** to find out if they can provide us any help with our project or give us any feedback. If you want to find out more about our Integrated Human Practices you can click [here](./human_practices). We also got in contact with a lot of **local stakeholders to talk about the relevance of our project or biofilms in general**.

We also had an amazing **talk with iGEM Monterrey from Mexico**, since both our teams are interested in biofilms. We were able to **share some ideas and scheduled another meeting**.

We participated in the **iGEM monument challenge** by iGEM Kaiserslautern and shared some of the most popular attractions of Darmstadt with the iGEM community.

In our **project development meeting** we made huge steps forward by **discussing the different approaches of the research groups** in detail. We got a **lot of inspirations** for our research in the next few weeks but also had to **discard some ideas**. Also, we got some feedback from our advisors, that we have to focus our research, so we had a little bit of **restructuration** planned for the following weeks.

Last but not least we **planted our team plant** together in our team meeting.

Week 17

This week, we had to **reschedule our weekly plan**, since we got the feedback that we need to **focus our research**, because we were running in too many directions with our research. We therefore cancelled our small group meetings this week to **define clear to-dos for our project**.

Also, some of our teammates contributed at the **Sustainable Research Symposium** and we got a lot of interesting information about the sustainability of our project. We got so inspired, that we wanted to contribute in this sustainability discussion by having our **“Greener Lab” workshop** and sharing some of the information from the symposium with researchers at our university.

We also had a **Biobrick workshop** this week, talking about the iGEM Biobrick standard, assembly methods and plasmid construction. This workshop was really **helpful for our experimental planning** and the general understanding of some of the iGEM parts.

## May

Week 18

To **refocus the aims of our project** we made a brainstorming week for the restructuration: We started by collecting the key elements, aims and goals of our project on a board and then discussed these points in the team. By this we wanted to work out the **key elements of our project** and focus completely on this part to have our project **actionable and fun** for the whole team. On Saturday we had breakfast together and further discussed the points on our board.

Our **team plant started growing really tall** so we had to move it to a new and nicer plant top.

Week 19

During the last week we collected **awesome ideas how to fix our little issue with our project direction** and condensed them to **3-4 main concepts** which we could continue with: The first idea was to implement a **second chromosome into a *E. coli*** to produce a broad variety of **different phages induced by different signaling factors**, utilizing the high capacity of a second chromosome. Alternatively, we could use a **similar system in our original host organism *B. subtilis*** to have an immune like response to different invading pathogens. This system could even be used in combination with our last year’s project. As a second idea we thought of **phages for the food industry** as an **alternative for harmful chemicals** as cleaning agents for biofilms. We also talked about one of the ideas from our original research phase, using **phages to introduce siRNA systems to gut bacteria of honey bees** to protect them from invading mites. Lastly, we had the idea of implementing a system for the **directed evolution of bacteriophages** for example for an extended host range, extreme conditions such as pH or temperature and many more.

We decided to go for a **final voting** between the **sleeper cell in *B. subtilis*** detecting and killing invading pathogens and the **phage directed evolution** in our next team meeting to then continue with our research.

We had an amazing **workshop on science communication** held by **Sascha from the science birds**. We learned a lot about science slams, science communication in general, our role as young scientists and what we can do to contribute to a better understanding of science in society. See our [**wiki section about the workshop**](./communication) and also check out the science bird’s website if you are interested!

Our team was looking forward towards joining the **first official iGEM event**: The **iGEM opening festival**. We had an amazing time listening to interesting calls, having discussions with other scientist from the field of synthetic biology and taking about the iGEM experiences and projects of other teams! With this meeting, out team **really caught the iGEM spirit and was motivated to work even harder** for our project.

Week 20

We talked about the two directions for our project again and had our **final vote**: We decided to continue with the **sleeper cells**! We were really happy to have new goals set for the following research phase.

We also recapped the **iGEM opening weekend**, which was an amazing experience for us and a lot of fun!

Week 21

We continued with our research groups and **shifted some of the topics** because of our project restructuration: The **biosafety group** became the **kill-switch group** developing a kill-switch triggered by QS signals and the **detection group** continued with research about ***B. subtilis* biofilms**. We had to read a lot of new literature and thus were pretty busy.

To develop our project and find proof of concept experiments, we talked a lot about the **DBTL cycle** which we would implement into our planning.

## June

Week 22

This week we were looking for **applications of our project to include into our Modeling work**.

We also **finished the script for our first podcast episode**, planned as an introduction of our team and synthetic biology in general.

We also contacted our uni’s **INSPIRED team**, a project about the colonialization of mars with an extraterrestrial greenhouse, to have a **meeting together and talk about our projects**.

We also talked about the **project promotion video**, which would be due in July and started a group responsible for the script.

For our **wiki** we started with a **framework to put our texts in**, to be as ready as possible for the wiki freeze which always comes sooner than expected.

Week 23

This week we really focused on **collaborations with other iGEM Teams**: We **met with iGEM Team Aachen** for our iJet collaboration, we scheduled our **next meeting with iGEM Monterrey**, designed our **postcards** for the postcard collaboration of **iGEM Düsseldorf**. We also met with the team from **Bochum** to talk about our projects and share some ideas together. In our first meeting with the **iGEM Team from Chicago** we talked about the **model of our project B-TOX from 2020** and also got some feedback regarding our project, especially the **genetic circuit modeling**.

We also talked to **iGEM Turku from Finnland** about laccases, since we focused on them last year and they were also working with microbial laccases this year.

We prepared our applications for the **iGEM safety and impact grants**, lucky to get these great possibilities of funding for our project.

We collected **name ideas** for our project that we wanted to set till next week.

Our team wrote an article for the biotech and biology journal **“Biospektrum”** to introduce our iGEM Team to the german bio community.

Together with **Stuttgart’s iGEM Team** we finished this week with an **amazing game night**.

Week 24

We had an **extended team meeting** this week intensively **discussing our progress** with our advisors and the team. We got a lot of **amazing feedback** and made some really **good progress** in this meeting.

Our **project promotion video team** came up with a **script for our video** and searched for actors playing detectives and phages in our video. If you want to check the video out, just follow [this link](https://video.igem.org/w/tcDrd5NsxH9ZNLZddPCRpo).

For some socializing in the team we had a **pub quiz** online together, which was really fun!

Week 25

This week we got the first results from our ***in-silico* SnapGene planning**. We **finished the filming of our project promotion video** and went ahead with the video production and some animations. We also talked a little bit about the **story of our project** and the **application possibilities** of our system.

We also were told, that it **may be possible to enter the lab in August**! We were really stoked about these news and focused on planning simple proof of concept experiments to be as productive as possible in the lab.

In this week we also had a **project development meeting** on Saturday, where all the small groups updated us about their progress. A lot of groups presented their **SnapGene constructs and POC experiments**, so this meeting really helped us on our way into the lab! Besides that, we talked about the **medal criteria** that we want to achieve and their current status in our team.

Week 26

We talked a lot about our **parts** and how we would order them to get ready for the lab as soon as possible. We decided to go for type IIs restriction enzymes and **Golden Gate assemblies** for our cloning because of their reliability and easy implementation.

For our **project presentation video** we were starting with our **video editing** and **animations**.

Another important point we talked about was the **selection of our iGEM track**: We discussed all tracks and old iGEM projects from these tracks with the team to raise our awareness for the track selection.

## July

Week 27

This week we had a long team meeting intensively **discussing our progress on the main research topics**:

For the **implementation of phages into a *B. subtilis* biofilm** we discussed how we could **mimic the specifically adapted expression system of bacteriophages** in their **host organism** in our sleeper cells.

We had a **major breakthrough** in our **research about biofilms** with an expert interview with Dr. Bischofs-Pfeifer and Prof. Dr. Kovács: They recommended us **strains for the formation of biofilms** and told us that **homogenous biofilms** of different genotypes might already be possible without metabolic dependence etc.

We **finalized the constructs for our kill-switch** proof of concept experiments and started the planing of our **pathogen sensing genetic circuit**.

For our Modeling we decided to write a **software** to **combine two models for metabolic dependency** of two bacterial strains in a single interface. Additionally, we started modeling the **response of our pathogen sensing circuits to AHLs** to improve our future lab experiments in this part of the project.

In **Human Practices** we **continued our podcast**, worked in education by writing **audiobooks for our Tonie-Boxes**, talked to a lot of experts about **ethics and policies** and started planning a **science slam** for the german iGEM community.

Week 28

This week we talked about the **design of our wiki page**. We talked a lot about **inclusivity** and how we can make the wiki as **barrier-free** as possible. We also decided to have a **“dark-mode” design** and created a document, were we wrote down all the design principles for our wiki.

We were all excited to design our **team hoodies**, which turned out really nice in the end. We wanted to get **fair-trade and sustainable merchandise** for our team.

We also discussed the **progress in our project research, HP and modeling** since our last meeting on monday really intensively. Unfortunately, we also realized that the **science slam** can most likely **not be hosted by our team**, since not that many teams agreed to prepare a short slam for the meeting.

Week 29

This week we decided to pick **New Application** as our track for the iGEM competition. We had a vote based on the **discussions** and our **Miro board** regarding the track selection from the previous weeks. We were really happy with this choice and **eager to further refine our project**.

We also prepared a **presentation** about our project held in front of our **uni’s faculty**. By this we were looking forward to get some valuable feedback and input for our project.

Week 30

We came up with a **novel genetic circuit** for a proof of concept of **lambda phage induction** in *E. coli* using the native repressor/regulator systems **cI and Cro**. As a first readout a **fluorescence measurement** is supposed to be conducted but phage expression in *E. coli* cells with an integrated lambda prophage. We finished the **second episode of our podcast**, which we were really proud of!

We also were able to order **Tonie-Boxes**, which we could use in kindergartens to teach children **basic scientific knowledge** as part of our HP education work.

In our **faculty presentation** we presented our project to our uni’s biology faculty and experts of our team that we invited. We were able to give an **awesome talk** to all participants and get a **lot of feedback** regarding our project. We also decided to have a **second faculty presentation** after finishing our project presentation video to prepare for the judging session.

## August

Week 31

This week we had a talk with our PI **about the lab work**. We were able to **get into the lab** but only under **strong conditions** due to the pandemic. We worked out a **hygiene concept** to give as many members of our team as possible the ability to work in the lab and get some experience. Some of them haven’t been in the lab since they started university so it was a **great opportunity** for all!

For the experiments in the lab, we decided to **shift all experiments to *E. coli*** as host organism. In agreement with our advisors and PIs it seemed to be the best possibility to **provide proof of concept experiments** in this short time in the lab with varying lab members each week. This meant a lot of **replanning to adapt all experiments to *E. coli*** for the next parts, to get ready to order our parts and begin with the lab work. We planned our assemblies using the **GoldenBraid system**, since its already established at our university.

We also discussed a lot of details about our iGEM lab with our team. In our hygiene concept we decided to have **weekly Covid tests** and to have **four people simultaneously in the lab**. We talked about the devices and instruments we have in the lab and the assembly plans for our team. We also had small groups that each prepared **protocols for the experiments** we had planned adapted to the materials we had in the lab.

Week 32

This week we were more than happy and really honored to **receive the iGEM Team Grant**.

We made **plans for our lab schedule** and organized our members for the several lab weeks, so **everybody gets the experience to enter the lab** at least once.

We presented the **workflow and cloning plan** for our first lab week using GoldenBraid again to the whole team and discussed it in detail.

To prepare for our **project presentation video** we scheduled **presentation trainings** for the whole team held by experienced ex-iGEMers from TU Darmstadt.

For our **wiki** we talked about the **structure** again and started to make the plans for all topics of our project. We discussed how we wanted to present our research, results and discussions and what pages we need for that.

We also had the **interview with molecular cloud**, check it out if you want to learn more about our team and our project.

For our final wiki phase we decided to have at least 4 weekends blocked for the whole team to **work on our wiki together**. We were really excited to finally present all our results to the iGEM community and collect everything we learned so far.

Week 33

We started this week with our **safety training** for our team, so everybody would be able to enter the lab starting from this week. We learned a lot about **safety in the lab**, **general safety** regarding GMOs and **good laboratory practice**.

We spoke about our assays in detail and came up with **well-plate schemes** for our assays that could be done within the next few weeks.

Akos T. Kovacs kindly provided us ***B. subtilis* strains for biofilm formation**. We wanted to use these **fluorescence labeled cells** to conduct first experiments regarding the **homogeneity of our biofilms**.

We continued to **finish more podcast episodes** and **finished our survey about synthetic biology**. Also, we prepared **workshops for high school students** in the next few weeks.

For our lab we **transferred our materials and consumables into our iGEM lab** and organized everything for the upcoming lab work. To organize everything and save our lab results or protocols we created a **detailed notion page** containing our **lab inventory and** a **digital lab book**.

We had the first **presentation trainings** this week with a lot of fun presentations. We were able to learn a lot and really **improved our presentation skills**. This would be especially useful for the final presentation video.

Week 34

We implemented our **quorum sensing based *Pseudomonas aeruginosa* sensing circuit** in a Python model. Additionally, we thought about adding a second **module calculating the expected phage expression** based on the AHL input generated by our circuit. For the model we discussed in the whole team how we could get **parameters for our model** and came up with a few promising solutions for that problem.

In the **lab** we prepared **cryo-stocks of our biofilm forming *B. subtilis* strains**, carried out a first **golden gate test reaction** and **grew our first biofilms** for our microscopy measurements of *B. subtilis* mKate GFP cocultures. We also **tested our equipment** with PCR reactions, agarose gels and first golden gate assemblies of test parts.

We talked about the **design of our wiki** and started with the **first design of figures** for our wiki.

Also, we **ordered the T-Shirts and Hoodies** for our team, which we were really looking forward to in the last weeks.

## September

Week 35

This week we were excited to **discuss our first results from the lab**: We made **fluorescence microscopy pictures** of the biofilm monocultures and also established a **first coculture** of two differently marked *B. subtilis* transformants.

Also our **parts arrived** and we started the first **Golden Gate assemblies of our QS based *P. aeruginos*a sensing genetic circuit**. These assemblies would result in our first **⍺-level plasmids** of our GoldenBraid approach, which then could later be combined to form **𝝮-level plasmids** with our completed construct.

For our final wiki phase we scheduled **wiki weekends beginning from the 11th of September**. With these weekend meetings we wanted to gather the whole team (digitally) to productively work on our wiki texts together.

We began to film a **short team introduction video for the promega instagram page**. We wanted to introduce our research foci and explain our project for the broad audience of the instagram channel.

Week 36

This week we mainly focused on the **first of our wiki weekends**. We **planned all texts** we wanted to write for our wiki and distributed them to different authors from our team. On the first day of our wiki weekend we first met at a local park to **take some photos** for our wiki and then went home to start the writing and correction process.

We also finished the short introduction of each our team members for the [promega video (external)](https://www.instagram.com/p/CUWz610j0WG/).

Additionally, we **talked about the figures** we wanted to include in our wiki. Since we **focused on inclusion** this year, we had to make sure they are **easy to understand** and that the **colors are distinguishable** for color blind people. If you want to find out more about that, **check out our guide on [inclusivity](./inclusivity)** for websites, such as our wiki.

In the lab we continued with the **assembly and sequencing of our first 𝝮-level plasmids**. We had some trouble with some of the constructs and thus had to repeat some of the assemblies.

Week 37

One of our team members prepared an **introduction into scientific writing**, which we all profited from for our wiki texts. We further discussed how we wanted to structure our wiki pages and how we want to include our figures.

We also wrote an **algorithm to analyze the appearance of biofilms in iGEM wikis** within the last few years. We found out, that this topic gets **increasingly relevant** for the synthetic biology community, which also highlights the **importance of our project** introducing an **additional safety layer** to these systems.

In the lab we made first **fluorescence measurements** with the cloned **𝝮-level plasmids** in our plate reader, but unfortunately **no fluorescence was detected** upon induction. We thus focused on the **troubleshooting** of our assays to figure out potential issues during the measurements.

Week 38

We still encountered **problems with the IPTG induction of our BL21(DE3) cells** and put a lot of focus on fixing this problem. We also worked on the **safety data sheets** of *E.* *coli* cells we got send from Prof. Dr. Węgrzyn to eventually use *E. coli* cells containing the lambda prophage for the **measurement of our genetic circuit containing *cI* and a *recA* mutant** for induction of its lytic state.

In the lab we focused on the **assembly of the remaining 𝝮-level plasmids** and the **sequence verification** of the assembled ones by both restriction digests and Sanger sequencing.

Week 39

This week **all genetic parts for our project arrived in our lab** and we started all assembly reactions for our plasmids. Dr. Rapp from our university helped us with the **fluorescence microscopy** of our biofilms and we got some **amazing shots** of our cocultures.

We also talked about **upcoming deadlines** and recapped the collabos we had within this iGEM year.

On our **wiki weekend** we wrote a lot of texts, created figures and continued with the creation of our website. We worked really productively and got a lot of texts done this week.

## October

Week 40

We successfully **cloned the Cre recombinase** for our kill-switch assays into a production plasmid. We also managed to **fix the problem** we encountered with the **IPTG induction of the T7 polymerase in BL21(DE3)**. The **genetic circuit for our lambda phage lytic cycle** induction was also started to get assembled, but we encountered some problems with this multiple-fragment golden gate reaction. Nevertheless, we got the **plasmid assembled** at the end of the week and were ready to **measure all our constructs in the next week**, which was really exciting for us.

We also wrote the **script for our project presentation video** and started to look for **locations** for us to film at. Like always in this final phase of iGEM we also talked about upcoming deadlines for the team and the challenges we still had in front of us.

Week 41

This week we **successfully conducted the assays for the genetic circuit** designed for the proof of concept of our phage induction construct. We were able to detect green fluorescence upon IPTG induction caused by the expression of *recA730*. For all other genetic constructs, we were unfortunately not able to measure our expected outputs. We were unable to perform troubleshooting on these constructs due to the lack of time with the wiki freeze ahead.

We were able to **finalize our wiki** on the last wiki weekend, finishing our wiki texts and figures. We also **started filming our project presentation video**.

Week 42

This week the **final wiki freeze** would take place. We were all excited to submit our wiki and still worked hard on our project presentation video.

- [February](#february)
- [March](#march)
- [April](#april)
- [May](#may)
- [June](#june)
- [July](#july)
- [August](#august)
- [September](#september)
- [October](#october)

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Contact us

igem@bio.tu-darmstadt.de

About us

We are the 2021 iGEM Team of the TU Darmstadt. This year
we focused on creating a self-protecting biofilm based on
*Bacillus subtilis* "sleeper cells".

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# Team:TU Darmstadt/engineering

Engineering – TUDA iGEM 2021

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Primary Menu

* [Home](./)
* Project
  + [Description](./description)
  + [Biofilm](./biofilm)
  + [Pathogen Sensing](./sensing)
  + [Bacteriophages](./phages)
  + [Biosafety](./biosafety)
  + [Contribution](./contribution)
  + [Engineering](./engineering)
  + [Proof of concept](./proof_of_concept)
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# Engineering

We conceptualized and started creating a multi-functional *Bacillus* *subtilis* biofilm which detects the pathogen *Pseudomonas* *aeruginosa* and in turn activates the expression of a bacteriophage which specifically kills the respective pathogen. Additionally, we developed a kill-switch to prevent the escape of viable, engineered cells from the biofilm.

We here illustrate the process of engineering and highlight the challenges we faced. We implemented the **Design-Build-Test-Learn (DBTL) cycle** to overcome these challenges and develop new solutions. **Practically,** we were not able to go past the first iteration of the DBTL cycle, due to the lack of time in the laboratory because of [COVID-19 measures](https://2021.igem.org/Team%3ATU_Darmstadt/safety#covsav). **Theoretically**, however, we elaborated multiple iterations. In this way, we gradually came closer to our final goal of a safer, self-defending biofilm.

## Pathogen sensing

### Pathogen Sensing via Transcriptional Regulation

We use two **allosteric transcription factors (aTF)**, which originate from the quorum sensing system of *Pseudomonas* *aeruginosa* to create a sensing circuit in ***Bacillus*** ***subtilis*** **sleeper** **cell** (“sleeper state” until being activated). This circuit responds to the **presence of *P.*** ***aeruginosa***, **a common pathogen**, with the production of a **bacteriophage which specifically kills *P.*** ***aeruginosa.***

It has been shown that *P.* *aeruginosa* cells can be sensed using either the aTFs [QscR](http://parts.igem.org/Part%3ABBa_K3985003) or [LasR](http://parts.igem.org/Part%3ABBa_K3985001).​1,2​ LasR is a transcription factor, which regulates the expression of genes involved in quorum sensing by binding the promoter *PLux*.​3​ QscR is a quorum sensing repressor, which can inhibit the function of LasR. Besides this, QscR is also able to control gene expression directly by binding the promoter *PPA1897* thus acting as a transcription factor.​4​ Both aTFs bind the acyl homoserine lactone (AHL) *N-(3-oxododecanoyl)-homoserine* *lactone* (**3OC12-HSL**) which is a quorum sensing autoinducer native to *P.* *aeruginosa*.​3,4​ The respective aTF and 3OC12-HSL form a complex that binds to its respective **inducible promoter.** In our circuit, the aTFs are expressed constitutively to control the expression of *recA730* via the respective inducible promoter ([Figure 1](https://2021.igem.org/Team%3ATU_Darmstadt/engineering#recacuircit)). The protein **RecA730** is responsible for the switch from the lysogenic to the lytic cycle of the phage and thus for controlled **phage production**.​5,6​

![](/wiki/images/4/4b/T--TU_Darmstadt--Regulon_Phage_output_1910-1024x578.png)

To prove the inducibility of our designed circuit, we exchange *recA730* with [*egfp*](http://parts.igem.org/Part%3ABBa_K3985005) ([Figure 2](https://2021.igem.org/Team%3ATU_Darmstadt/engineering#egfpcuircit)). If the circuit works properly, we will obtain a **green fluorescence signal as an output**. Wu et al. (2021) and Saide et al. (2011) have already successfully detected *P.* *aeruginosa* using *E.* *coli* by expressing *egfp* in the presence of 3OC12-HSL.​1,2​

![](/wiki/images/6/66/T--TU_Darmstadt--Regulon_GFP_output_1910-1024x578.png)

Since this circuit has not yet been evaluated in *B.* *subtilis,* we planned to use a **DBTL** cycle to generate a functional sensing circuit. Although we have been able to assemble the genetic circuit we can not present experimental data of this project part since our time in the lab was limited. Therefore, we performed a theoretical DBTL cycle.

First of all, we want to characterize the **constitutive promoter** *Pveg* of ***B.* *subtilis****.* Therefore, we express the red fluorescent protein **mKATE2** under its control and measurethe **fluorescence intensity over time.** This way we characterize the used promoter within a [fluorescence assay](./experiments). The possible results are either a measurable fluorescence or none.

* If we measure a fluorescence signal, it means the constitutive promoter *Pveg* is active and mKATE2 is functionally expressed*.* From this result, we assume that the gene encoding the aTF will also be transcribed. This does not yet ensure functional translation and folding of the aTF.

* If we do not measure a fluorescence output, mKATE2 is not functionally expressed and it is unlikely that another gene will be functionally expressed under the control of the same promoter and the same RBS. To solve this problem one option would be to **exchange the constitutive promoter** to ensure functional transcription:Besides *Pveg*, we also plan to test *PliaG* and *PlepA* originating from *B.* *subtilis.* Another option is to **vary the RBS** to ensure functional translation. We plan to use a consensus [RBS](http://parts.igem.org/Part%3ABBa_K090505) for *B.* *subtilis* from the iGEM parts registry as well as a calculated RBS, obtained from the Salis Lab RBS Calculator.​7​ Varying the promoter and RBS allows us to identify the most promising combination for the tested parts.

The second step in our test procedure is to investigate whether the **inducible promoters** *PPA1897* and *PLux*, which were previously used in *E.* *coli,* are **functional in *B.* *subtilis***.​1,2​ Therefore, we put *egfp* under the control of the respective promoters and constitutively express the aTFs QscR or LasR. We then measure the **green fluorescence output** in the presence of 3OC12-HSL. We **vary 3OC12-HSL concentrations** between 10-4 M and 10-10 M​1,2​ to assess the **sensitivity** of the sensing circuit. Besides the inducibility, we also investigate on the question whether 3OC12-HSL passes the Gram-positive cell barrier of *B.* *subtilis.* This experimental design was recommended by [Prof. Dr. Anke Becker](https://2021.igem.org/Team%3ATU_Darmstadt/human_practices#pathsens) to test if the AHL enters *B.* *subtilis* cells.

If we do not measure a green fluorescence output in this experiment, either the aTFs are not functional in *B.* *subtilis* or the AHL cannot enter the cell.

An option to find out if the aTFs are functional in *B.* *subtilis* is to generate a **cell free extract from *B.* *subtilis***. With this third experiment, the **function of the aTF** in the presence of 3OC12-HSL is tested without the cell barrier.​8​ This can either result in a green fluorescence output or not.

* If we do not measure a fluorescence output, the aTFs are probably not functional in *B.* *subtilis* cells.​8​ This means we would have to adapt our sensing circuit to establish a functional system in *B.* *subtilis*.

* If we measure a fluorescence output our sensing circuit is working. Consequently, we then have to prove that the AHL can enter *B.* *subtilis* cells.

To test if the **AHL can pass through the cell barrier**, we need a fourth experimental design. A possible experiment is to add AHL to the medium of a *B.* *subtilis* culture and **measure the remaining AHL concentration** in the medium over time. This measurement could be carried out with **analytical methods** like mass spectroscopy or gas chromatography. From this experiment, we can expect two outcomes:

* If the AHL concentration in the medium **decreases** over time, the AHL **enters** the *B.* *subtilis* cells.

* If the concentration of the AHL remains **constant**, the AHL does not enter the *B.* *subtilis* cell. In that case our design for the sensing circuit in *B.* *subtilis* would have **failed**.

Nevertheless, we have vastly researched literature on this topic. [Prof. Dr. Anke Becker](./human_practices#pathsens) assumed that AHL molecules should be able to diffuse freely through the cell membrane, as they are small hydrophobic molecules. Additionally, it has been reported that AHL can regulate the gene expression of other Gram-positive bacteria like *S.* *aureus* or *L.* *monocytogenes.*​9,10​ This **indicates** that our fourth **experiment likely turns out positive**. A more detailed explanation of our research results can be found in the section [*N-Acyl* *homoserine* *lactones* and *B.* *subtilis*](https://2021.igem.org/Team%3ATU_Darmstadt/sensing-background#emnemacylhomoserinelactonesandembsubtilisem).

After showing functionality of our sensing circuit in *B. subtilis*, the last steps would be to exchange *egfp* with *recA730* and to test if **RecA730 is synthesized in the presence of 3OC12-HSL**. Therefore, we would fuse RecA730 with a His6-Tag and detect the expressed protein using anti-His antibodies in a Western Blot. We here expect a band at about 38 kDa​6​ to confirm the expression of RecA730.​1​

If this works, we combine the sensing circuit with the **genetic switch which shifts the phage** from the lysogenic to the lytic cycle. This switch is further explained in the next section.

## Bac­terio­phages

### Phage Induction and Release in Non-host Organisms

[Our genetic switch](./phages#phageinduction) is designed to activate phage induction when a quorum sensing molecule from an invading pathogen is detected. We therefore utilize a native mechanism for the induction of lysogenic prophages to lytic phages. For our proof of concept lambda phage induction the production of **Cro is necessary**. Since *cro* is regulated by the ***PR* promoter** our genetic switch aims to **diminish the lambda repressor cI** which represses *PR*. The basic concept of our switch is based on three proteins, where **RecA730** and **TetR** act as **antagonists** to the lambda repressor cI. The produced TetR binds to the *PtetO*promoter and herewith prevents production ofcI. The produced RecA730 cleaves cI, whereby the *PR* promoter is no longer repressed and ***cro* is expressed**, thus leading to the **activation** of the **lytic cycle**.

As proof of concept we want to show that our genetic switch is able to activate the *PR* promoter by deactivation of cI. Therefore, we used the **fluorescent protein eGFP** as a reporter under the control of the *PR* promoter. Also, the genes for *recA730* and t*etR* are regulated by a *T7* promoter. Using *E. coli* BL21 (DE3) the functionality of our genetic switch can then be evaluated through **IPTG induction**. An overview of our genetic switch can be seen in [Figure 3](#phageinduction). After a successful proof of concept this genetic switch can be expanded through combination with the genetic circuit for Pathogen Sensing and implemented into *Bacillus subtilis*.

![](/wiki/images/1/12/T--TU_Darmstadt--Phagies-GenCirc_Ecoli_readyforupload.svg)

Figure 3. **Schematic overview illustrating the design of our genetic circuit for phage induction. a.** General order of the genetic elements in our construct. **b**. Our genetic circuit before induction through IPTG: repression of the *egfp* expression through expression of *cI*, which is representative for the phage being kept in lysogenic state. **c.** Our genetic circuit after induction with IPTG: expression of *recA730*, cleaving cI and expression of *tetR*, which represses the expression of *cI*. This leads to eGFP production which is representative for the phage switching to lytic state.

#### RecA730 and TetR

Testing of our [genetic switch](http://parts.igem.org/Part%3ABBa_K3985026) was a **great success**. We were able to detect a **12-fold increase of eGFP** upon induction compared to no induction. See our [results](./phages#lab) here.

Since the controlled production of eGFP worked as expected the next step would be testing the induction of the lysogen. Unfortunately, we were not able to test this in the lab and will develop from here on a theoretical DBTL Cycle.

There are some **considerable differences to the [native switch](./phages#native)** in the lysogen. The lambda repressor *cI* is constitutively expressed in cells containing the lambda prophage, resulting in higher cI concentrations than in our measurements. Eventually, **RecA730 activity could be too low** to cause efficient deactivation of the repressor resulting in reduced reporter output. This effect could be compensated by either changing regulatory domains for increased RecA730 levels or an knockdown/knockout of the *cI* gene on the prophage. Necessity of these adaptions could be assessed in identical fluorescence and optical density measurements carried out in the lysogen.

#### Combination with Pathogen Sensing

If the controlled induction of the lysogen is achieved, **the quorum sensing (QS) based switch** needs to be integrated into the induction module of our construct. The two proteins RecA730 and TetR are then regulated by the quorum sensing sensitive promoter [*PPA1897*](./sensing-circuit). Also, the allosteric transcription factor QscR needs to be expressed constitutively.

This construct can then be tested with the [previously established fluorescence assay](./proof_of_concept#pathogensensing). If the quorum sensing controlled degradation of cI is verified again, a similar experiment shall be conducted with the lysogen. This time the optical density should decrease 45 min upon the culture has been exposed to the QS signal.​5​

#### Controlled Lytic Cycle Induction and Phage Release in *B. subtilis*

For the implementation of this system in *B. subtilis* the **phage assembly must be achieved**. The highly adapted expression systems evolved in *E. coli* could be transferred to *B. subtilis* by **variations of regulatory elements and codon optimization**. The system responsible for timed cell lysis for the [phage release](./phages#release) must also be adapted for the new host organism. This could be achieved by protein engineering of holines, membrane pores for cell lysis. Alternatively, transcription and translation of these holines could be tuned yielding in the desired delay between pathogen sensing and lysis. For integration of the phages, we are proposing a [phage integration site](./phages#integration) similar to systems inducing the lysogenic state in lambda phages.

To **adapt our system to specifically lyse pathogens**, the genetic switch must be adapted to regulatory systems of respective bacteriophages targeting the desired pathogens. The implementation of the lambda regulatory system displays a proof of concept for the inclusion of lysogenic-lytic regulatory systems of prophages. This highlights the feasibility to extend a similar approach to multiple lysogenic phages, matching our idea of a modular approach adaptable to a multitude of phages.

We do recognize there are still many major challenges to achieve the complete synthesis and assembly of functional phages in a non-host organism. However, we think these are helpful and necessary steps to overcome the remaining hurdles through an iterative approach in the form of a DBTL cycle.

## Biosafety

### Inversion via Cre-Lox Recombination

As we further developed the kill-switch design of last year’s iGEM Team of TU Darmstadt, we generally optimized the schematic framework by adding RBS and terminators to enable transcription and translation. As previously mentioned in [Adaptation of Parts](https://2021.igem.org/Team%3ATU_Darmstadt/biosafety#adapecoli), we considered required changes within our construct to adapt to *E.* *coli* as new chassis. Thus, the kill-switch design was fitting to conduct potential assays in *B.* *subtilis* as well as *E.* *coli*.

In case of our [proof of concept](https://2021.igem.org/Team%3ATU_Darmstadt/proof_of_concept#creloxpoc) assay turns out to be successful in *E.* *coli*, we can assume that, due to the **conserved lox sites**, the inversion of the **final cassette** should be **functional** in ***B.*** ***subtilis*** as well. Nevertheless, we are aware that information and findings gathered in *E.* *coli* cannot be directly transferred to *B.* *subtilis* since we switch between two different organisms which differ in several aspects, e.g., the membrane architecture [from Gram-negative *E.* *coli* to Gram-positive *B.* *subtilis*)*.* Additionally, *E.* *coli* is lacking the two-component ComQXPA-system.​11​ Hence, we need an extended proof-of-concept assay to test whether the ComX signaling is functional in *B.* *subtilis*. This could be achieved in a DBTL process based on the framework of our current genetic circuit for *B.* *subtilis*. If meeting this requirement as well, the final kill-switch cassette would be eventually implemented in the biofilm forming strain DK1042. Functional ComX can be heterologously produced in *E.* *coli.*​12,13​ Recombinant ComX can then be added externally to the system to induce expression of the essential gene *rpsB*. As mentioned in the previous section [Theoretical Elaboration: *B.* *subtilis*](https://2021.igem.org/Team%3ATU_Darmstadt/biosafety#theobsub), the final cassette design ([Assay 4](https://2021.igem.org/Team%3ATU_Darmstadt/biosafety#assayfourtoggle)) contains a bidirectional promoter cassette flanked by *lox66* and *lox71* with constitutive *Pveg* and QS-inducible *PdegQ* promoter. After Cre-Lox inversion of the cassette, expression of the essential gene should be induced by the **transcription factor ComA-P**.​14​

Since the **functionality** of our final kill-switch fully **relies on** the **ComQXPA-system**, we are aware of the fact that our engineered kill-switch cassette up to this point might not be optimal for implementation in our biofilm. To address potential weaknesses several parameters can be adjusted to yield an optimized kill-switch.
We could face the issue that the **cells** are **not viable** under **transcriptional control of *PdegQ****.* Cell death can be detected via Live/Dead staining. Since *PdegQ* is a non-native promoter for the *rpsB* gene, the transcription efficiency might be too **low** to achieve a sufficient **expression level of *rpsB***. In this case, we could try to **optimize** the **binding affinity** of the RNA-polymerase by **modulating** the **operator region** upstream of the promoter core motif of *PdegQ*.​15​

On the translation level, we could take **RBS** **shuffling** into account. Thus, we would be able to adapt spacing between the core Shine-Dalgarno motif and either start codon or *PdegQ* promoter​16​, while not affecting the –35 and –10 regions. This way, we would try to improve translational efficiency.

Adding a **N-terminal His-tag** to recombinant proteins can improve expression levels.​17,18​ In this case, we would test if *rpsB* further maintains proper function with addition of a N-terminal His-tag.

Furthermore, it would also be an option to **replace** the **essential gene**. Doing this, we would still focus on **translational control** since replication is not constantly needed and thus not that **critical for** the **survival** of the cell in the first place. Choosing the essential gene is challenging, as it is way easier to control a gene separately than within an operon containing several regulatory genes.
A potential alternative would be ***thrS*** coding for the threonyl-tRNA synthetase, being part of a regulon. But its **paralogue *thrZ*** must be **knocked out** additionally for cell viability control.​19​

Given the fact that the kill-switch is completely functional within sporulation-deficient *B.* *subtilis* *168*​20​, we could further **optimize** the **chassis** and try to implement our system in a *B.* *subtilis* strain with a **minimal genome**.​21,22​ Doing this, we would have to make sure that essential genes for biofilm formation are not excluded. Thus, we could minimize potential interactions of our kill-switch system and make it more independent from environmental conditions, thereby positively affecting **biocontainment efficiency**.

All in all, **empirical optimization**, including multiple DBTL-iterations, will be **required to engineer** the **optimal kill-switch**, thus ensuring biocontainment of our system for application outside of the lab. This way, our genetic construct could provide a **reliable system** to prevent the uncontrolled release of GMOs **easily adaptable** to a multitude of biofilm based systems.

### References

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17. 17. Park W-J, You S-H, Choi H-A, Chu Y-J, Kim G-J. Over-expression of recombinant proteins with N-terminal His-tag via subcellular uneven distribution in Escherichia coli. Acta biochimica et biophysica Sinica. 2015;47:661. doi:[10.1093/abbs/gmv069](https://doi.org/10.1093/abbs/gmv069)
18. 18. Shilling PJ, Mirzadeh K, Cumming AJ, Widesheim M, Köck Z, Daley DO. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Communications biology. 2020;3:214. doi:[10.1038/s42003-020-0939-8](https://doi.org/10.1038/s42003-020-0939-8)
19. 19. Reu DR, Commichau FM, Gundlach J, Zhu B, Stülke J. The Blueprint of a Minimal Cell: MiniBacillus. Microbiology and molecular biology reviews : MMBR. 2016;80:955–987. doi:[10.1128/MMBR.00029-16](https://doi.org/10.1128/MMBR.00029-16)
20. 20. Špacapan M, Danevčič T, Štefanic P, Porter M, Stanley-Wall NR, Mandic-Mulec I. The ComX Quorum Sensing Peptide of Bacillus subtilis Affects Biofilm Formation Negatively and Sporulation Positively. Microorganisms. 2020;8. doi:[10.3390/microorganisms8081131](https://doi.org/10.3390/microorganisms8081131)
21. 21. Sung BH, Choe D, Kim SC, Cho B-K. Construction of a minimal genome as a chassis for synthetic biology. Essays in biochemistry. 2016;60:337–346. doi:[10.1042/EBC20160024](https://doi.org/10.1042/EBC20160024)
22. 22. Michalik S, Reder A, Richts B, Fahauer P, Mäder U, Pedreira T, Poehlein A, van Heel AJ, van Tilburg AY, Altenbuchner J, et al. The Bacillus subtilis Minimal Genome Compendium. ACS synthetic biology. 2021. doi:[10.1021/acssynbio.1c00339](https://doi.org/10.1021/acssynbio.1c00339)

- [Pathogen sensing](#pathogensensing)
- [Bac­terio­phages](#bacteriophages)
- [Biosafety](#biosafety)

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About us

We are the 2021 iGEM Team of the TU Darmstadt. This year
we focused on creating a self-protecting biofilm based on
*Bacillus subtilis* "sleeper cells".

[![Eppendorf](/wiki/images/9/95/T--TU_Darmstadt--Eppendorf.png)](https://www.eppendorf.com/US-en/)
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# Team:TU Darmstadt/experiments

Experiments – TUDA iGEM 2021

![](/wiki/images/8/89/T--TU_Darmstadt--Header_Export.svg)

Primary Menu

* [Home](./)
* Project
  + [Description](./description)
  + [Biofilm](./biofilm)
  + [Pathogen Sensing](./sensing)
  + [Bacteriophages](./phages)
  + [Biosafety](./biosafety)
  + [Contribution](./contribution)
  + [Engineering](./engineering)
  + [Proof of concept](./proof_of_concept)
* Team
  + [Team](./team)
  + [Collaborations](./collaborations)
  + [Attributions](./attributions)
  + [Sponsoring](./sponsoring)
* Human Practices
  + [Overview](./overview)
  + [Integrated](./human_practices)
  + [Education & Communication](./communication)
  + [Implementation](./implementation)
  + [Ethics & Policies](./ethics_policies)
* Modeling
  + [Genetic Circuit](./model)
  + [Phoenix](./phoenix)
  + [GoGEM](./software)
* Lab
  + [Results Summary](./results)
  + [Parts](./parts)
  + [Backbones](./backbones)
  + [Experiments](./experiments)
  + [Notebook](./notebook)
* Achievements
  + [Awards](./awards)
  + [Medal Criteria](./medal-criteria)
  + [Inclusion](./inclusivity)
  + [Safety & Security](./safety)
  + [Excellence](./excellence)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3985)
* [Plain Language](./plain-language)

# Experiments

## Laboratory Methods

We collected all methods and protocols in a document to allow all **future iGEM Teams** and other researchers to **reproduce the experiments we conducted** during our iGEM project. We **discuss the protocols in detail** to allow you to fully understand our thought processes behind the used methods. We really hope to contribute to the iGEM open source platform, providing relevant scientific data and its background to everyone in the SynBio community.

[![](/wiki/images/c/c5/T--TU_Darmstadt--Methods-Deckblatt-724x1024.png)](/wiki/images/6/6e/T--TU_Darmstadt--tu_darmstadt_methods_2021-1.pdf)

[Methodic\_Protocols\_and\_Material](/wiki/images/1/11/T--TU_Darmstadt--tu_darmstadt_methods_2021-2.pdf)[Download](/wiki/images/1/11/T--TU_Darmstadt--tu_darmstadt_methods_2021-2.pdf)

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Contact us

igem@bio.tu-darmstadt.de

About us

We are the 2021 iGEM Team of the TU Darmstadt. This year
we focused on creating a self-protecting biofilm based on
*Bacillus subtilis* "sleeper cells".

[![Eppendorf](/wiki/images/9/95/T--TU_Darmstadt--Eppendorf.png)](https://www.eppendorf.com/US-en/)
[![hilgenberg](/wiki/images/f/fe/T--TU_Darmstadt--Hilgenberg.png)](https://www.hilgenberg-gmbh.de/en/)
[![Zymo Research](/wiki/images/a/aa/T--TU_Darmstadt--Zymo.png)](https://www.zymoresearch.com)
[![New England Biolabs Inc.](/wiki/images/5/54/T--TU_Darmstadt--NEB.png)](https://international.neb.com)

[![IDT Integrated DNA Technologies](/wiki/images/8/80/T--TU_Darmstadt--IDT.png)](https://www.idtdna.com)
[![Snapgene](/wiki/images/e/e7/T--TU_Darmstadt--Snapgene.png)](https://www.snapgene.com)
[![Biebertaler Blutegelzucht](/wiki/images/4/4a/T--TU_Darmstadt--bbez.png)](https://blutegel.de/)
[![Promega](/wiki/images/d/de/T--TU_Darmstadt--Promega.png)](https://www.promega.com/)

[![DWK Life Sciences](/wiki/images/7/79/T--TU_Darmstadt--DWK.png)](https://www.dwk.com/)
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# Team:TU Darmstadt/results

Results Summary – TUDA iGEM 2021

![](/wiki/images/8/89/T--TU_Darmstadt--Header_Export.svg)

Primary Menu

* [Home](./)
* Project
  + [Description](./description)
  + [Biofilm](./biofilm)
  + [Pathogen Sensing](./sensing)
  + [Bacteriophages](./phages)
  + [Biosafety](./biosafety)
  + [Contribution](./contribution)
  + [Engineering](./engineering)
  + [Proof of concept](./proof_of_concept)
* Team
  + [Team](./team)
  + [Collaborations](./collaborations)
  + [Attributions](./attributions)
  + [Sponsoring](./sponsoring)
* Human Practices
  + [Overview](./overview)
  + [Integrated](./human_practices)
  + [Education & Communication](./communication)
  + [Implementation](./implementation)
  + [Ethics & Policies](./ethics_policies)
* Modeling
  + [Genetic Circuit](./model)
  + [Phoenix](./phoenix)
  + [GoGEM](./software)
* Lab
  + [Results Summary](./results)
  + [Parts](./parts)
  + [Backbones](./backbones)
  + [Experiments](./experiments)
  + [Notebook](./notebook)
* Achievements
  + [Awards](./awards)
  + [Medal Criteria](./medal-criteria)
  + [Inclusion](./inclusivity)
  + [Safety & Security](./safety)
  + [Excellence](./excellence)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3985)
* [Plain Language](./plain-language)

# Results Summary

In this section we want to briefly **summarize the results** we acquired during the time we had in the wet lab. We aimed to conduct a [proof of concept](./proof_of_concept) of our adaptive immune-like system for the protection of functionalized biofilms. Due to restrictions at our university caused by the ongoing pandemic, we had very limited lab access. On this page we only **present a short summary of our results**. If you want to find out more about the experiments planned for our separate research topics, have a look at the corresponding pages. ([Bacteriophages](./phages), [Biofilm](./biofilm), [Biosafety](./biosafety) and [Pathogen Sensing](./sensing))

We used **computational methods** to also acquire results for our project and guide the development of our methods and assays. Especially, our genetic circuit model for the sensing of pathogens helped us to fully understand the genetic circuit and adapt the assay design.

## Biofilm

To conduct a proof of concept for a [multi-functional biofilm](./biofilm#lab) containing bacteria carrying different genetic elements we **cocultured *B. subtilis* DK1042 cells with different fluorescence markers**. DK1042 is a strain that forms biofilms, thus the resulting biofilms were analyzed by fluorescence microscopy. We were able to create stable cocultures without the need for synthetically introduced dependencies between the different genotypes. These results displays a proof of concept for the integration of multiple bacteriophages inducible by different signaling molecules. Each signaling molecule in this connection could trigger expression of a phage specific to the invading pathogen species.

![](/wiki/images/7/72/T--TU_Darmstadt--Overview-Plate-Rapp-1.svg)

## Bacteriophages

Bacteriophages can stably integrate into the genome of bacteria as prophages and remain unactivated throughout multiple cell division cycles. The lytic stage is induced commonly by stress reactions such as DNA damage and the connected signaling cascades. This **induction of phages** from a stable, genomically integrated state towards the active, lytic stage is crucial for the activation of our sleeper cell. We therefore want to use these established, evolutionary developed systems and put them under the control of our pathogen sensing circuit. As a [proof of concept](./phages#lab) for these kinds of circuits we tested the **inducible control of RecA730**, a constitutively protease-active variant of the lytic state trigger of the lambda phage, and its effect on a cI controlled fluorescence marker. cI is a tight transcriptional repressor keeping the phage in its lysogenic cycle upon cleavage by RecA and thus deactivation.

![](/wiki/images/7/71/T--TU_Darmstadt--Floureszenzassay-mit-Fit-Induktionssignal-2.svg)

## Pathogen Sensing

We [conceptualized a genetic circuit](./sensing-circuit) for the **sensing of *P. aeruginosa*** by recognition of a quorum sensing molecule. We were able to **assemble this construct**, but unfortunately didn’t collect any data on the genetic circuit.

For the validation and further improvement of the genetic circuit we set up a [model describing the reporter output](./model) based on literature and computed parameters.

![](/wiki/images/b/bb/T--TU_Darmstadt--Genetic_circuit_QscR_done-2.svg)

Figure 3. **Genetic circuit for the sensing of AHL expresses *egfp* in the presence of AHL**. The activated transcriptional factor signaling molecule complex initiates transcription of the *PPA1897* promoter.

## Biosafety

To secure biosafety and biocontainment of our system, we planned to further **optimize the kill-switch** from iGEM TU Darmstadt 2020. To do so, we planned **[experiments for the assessment of the switch](./biosafety#assaydesigns)** and adapt the single components. We were able to assemble the first constructs for these measurements but again were unable to test them in the lab to due very limited access we got. The initial part for assessment was the **activation of the kill-switch by the irreversible inversion of a promoter cassette**. This part was supposed to be screened by fluorescence measurements of our genetic construct.

![](/wiki/images/1/1b/T--TU_Darmstadt--Subtisstuff_1.svg)

Figure 4. **Genetic circuit for the assessment of a Cre-mediated promoter cassette flip**. After the flip, a constitutive promoter initially controlling *sfgfp* expression is exchanged with a QS activated promoter sequence. The reporter gene is to be exchanged with a essential gene in the actual construct to couple cell viability to QS reporter concentrations only reached in biofilm cell communities.

- [Biofilm](#biofilm)
- [Bacteriophages](#bacteriophages)
- [Pathogen Sensing](#pathogensensing)
- [Biosafety](#biosafety)

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Contact us

igem@bio.tu-darmstadt.de

About us

We are the 2021 iGEM Team of the TU Darmstadt. This year
we focused on creating a self-protecting biofilm based on
*Bacillus subtilis* "sleeper cells".

[![Eppendorf](/wiki/images/9/95/T--TU_Darmstadt--Eppendorf.png)](https://www.eppendorf.com/US-en/)
[![hilgenberg](/wiki/images/f/fe/T--TU_Darmstadt--Hilgenberg.png)](https://www.hilgenberg-gmbh.de/en/)
[![Zymo Research](/wiki/images/a/aa/T--TU_Darmstadt--Zymo.png)](https://www.zymoresearch.com)
[![New England Biolabs Inc.](/wiki/images/5/54/T--TU_Darmstadt--NEB.png)](https://international.neb.com)

[![IDT Integrated DNA Technologies](/wiki/images/8/80/T--TU_Darmstadt--IDT.png)](https://www.idtdna.com)
[![Snapgene](/wiki/images/e/e7/T--TU_Darmstadt--Snapgene.png)](https://www.snapgene.com)
[![Biebertaler Blutegelzucht](/wiki/images/4/4a/T--TU_Darmstadt--bbez.png)](https://blutegel.de/)
[![Promega](/wiki/images/d/de/T--TU_Darmstadt--Promega.png)](https://www.promega.com/)

[![DWK Life Sciences](/wiki/images/7/79/T--TU_Darmstadt--DWK.png)](https://www.dwk.com/)
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