iGEM 2021 — Team Duesseldorf

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# Team:Duesseldorf/Notebook

Notebook | iGEM Team DD

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* Team

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* Parts

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* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

![background_image](https://static.igem.org/mediawiki/2021/5/53/T--Duesseldorf--img--mike-tinnion-3ym6i13Y9LU-unsplash.jpg)

# Notebook

---

##### Contents

## 12/29/2020

The new iGEM team met for the first time and organizational things like weekly meetings and means of communication were discussed. Expectations were explained and the iGEM year was roughly planned out.

## 01/05/2021

Every member presented a successful iGEM project from the past by different teams and reviewed what aspects made it successful. Further ideas for public engagement, education, and collaboration were brainstormed. Past aspects like a newsletter and the postcard project were talked about.

## 01/12/2021

Responsibilities like mail, social media, and sponsoring were distributed. The implementation of public engagement and education was debated. Research for possible project ideas started and possible project directions included plant diseases, enzyme therapy, and bioluminescence-based biomarkers.

## 01/19/2021

Principal investigators who are available were looked for. Four possible project ideas were formed and the members divided the team into four smaller groups and assigned themselves to a project idea to do more research.

## 01/26/2021

An internal cloning tutorial was organized for members who have not had any experience with it so far. Project ideas were being narrowed down. It was planned to contact professors whose research is connected to our possible project ideas. Furthermore, the social media team has planned content for the coming months.

## 02/02/2021

For the first time companies were being contacted about sponsoring. The current progress on project proposals was presented.

## 02/09/2021

A date was set for the professor meeting to present our project ideas so far. At this point, two ideas have been chosen: a plant-gene-editing toolbox and the production of medical supplies with keratin.

## 02/18/2021

The responsibility for writing articles for the newsletter was divided. The planning of the professor meeting, like means of communication and a presentation, was organized. Deadlines to start lab work were set.

## 02/22/2021

The professor meeting took place, in which the two project ideas were presented and the members received constructive advice to choose a project.

## 02/23/2021

After getting input from the professors, the advantages and disadvantages of each project were discussed, and finally, it was decided to go with plant pathogen detection. Furthermore, it was talked about how the project could be implemented and more research was planned on methods and host/pathogen interaction.

## 03/02/2020

It was debated how working as a team and distribution of responsibilities could be improved. The election of student leaders was organized. Actual offers from Macherey-Nagel as a sponsor were received. More organizational things like equipment for an opentron, registration fee, and implementation of integrated human practices. Features of the project were discussed in more detail, like possible detection methods, experts, and the audience. The innovative part of the project was decided: pathogen forecast.

## 03/09/2021

The application for a Promega sponsoring was completed. Deadlines for the organization of the postcard project were set and outreach to experts was organized. More research into proactive biomonitoring was done, i.e. model host, model organism. Detecting different stresses with drones by biomonitoring a field was considered. It was contemplated to generate aptamers with SELEX. The implementation of the project in the lab was planned. Furthermore, the advantages and disadvantages of previously researched methods were weighed.

## 03/10/2021

Meeting with iGEM Bonn

## 03/15/2021

During the PI meeting, expectations and further meetings were discussed. It was explained how the project idea has changed from the last meeting with all professors.

## 03/16/2021

The responsibilities of the student leaders were determined. A detailed lab plan was created. Targets for detection were determined. The method of detection was set to be a sandwich lateral flow rapid test. A side project was introduced, which aims to identify promoters that react to stress and use fluorescent reporter proteins.

## 03/23/2021

It was discussed which track to compete in and also possible collaboration. More detailed SELEX protocols were established.

## 03/30/2021

Money for the iGEM registration was granted and the team was able to register. Storytelling for the project was considered. Also, the members split up into teams to do research more efficiently.

## 03/31/2021

The meeting with Prof. Dr. Meltem Avci-Adali took place, in which the team was advised on the SELEX procedures

## 04/06/2021

A statement of costs for our needs in the lab was created. While waiting for necessary materials, the members got to know common methods used in labs. A detailed lab plan with protocols was created. It was also decided to use gold nanoparticles for the detection method.

## 04/13/2021

More research was done into the detection method with gold nanoparticles and mechanisms. The modeling team discussed ideas about a heatmap and SELEX optimization.

## 04/20/2021

A meeting with an associate from opentron was organized. The medal criteria were checked again to direct the project in a better direction. A meeting with local farmers was organized to introduce the project idea to them,

## 04/22/2021

The meeting with Prof. Dr. Alga Zuccaro took place.

## 04/27/2021

Alternative methods to protein expression in E. coli were discussed. Also, the advisors went over experiment designs.

## 04/05/2021

It was discussed how the project corresponds so far with the given medal criteria. Another professor meeting was planned to discuss the current status of the project.

## 05/11/2021

The planning for the first experiments was finished. Collaboration was discussed during a meeting with iGEM Aachen and iGEM Bonn. The idea for a panel discussion was first mentioned

## 05/12/2021

The meeting with Prof. Dr. Mayer took place and the team received advice on aspects that could possibly endanger the detection process.

## 05/18/2021

The name for the project was thought about. Safety instructions at the labs were attended.

## 05/25/2021

The approximate cost for each test was calculated. Feedback was received from Prof. Dr. Djamei about proteins that could be used. Also, feedback by Prof. Dr. Mahlein about drone usage reached the team.

## 06/01/2021

The name CereX was decided on. The final stages of the postcard project were organized.

## 06/08/2021

The identity of the project was discussed for the promotional video. Ideas for a logo were brainstormed. During the meeting with Dr. Sommer, the team went over the pitch and decided to go with early diagnostics and prevention of diseases.

## 06/15/2021

The design and layout of the wiki were thought about. First logo ideas were displayed.

## 06/22/2021

The expression of a protein that needs to be detected was successful. Final preparations for the professor meeting were done. Questions for the meeting with associates from Bayer were prepared and the safety form was finished.

## 06/23/2021

The professor meeting took place. The team received a lot of useful advice on how to proceed and tips for the implementation of IHP.

## 06/29/2021

A meeting with Prof. Dr. Voegele took place. The team learned that pathogen differentiation is more useful than stress detection. The first shooting for the promotional video was done.

## 07/06/2021

First designs for postcards were thought about. The fulfillment of the medal criteria for education and HP were thought about.

## 07/13/2021

A collaboration by iGEM Calgary was discussed.

## 07/20/2021

A presentation of the project on the social media channels of PromegaGermany was organized.

## 07/27/2021

The design for the postcard was finished

## 08/03/2021

New protocols for cellSELEX were created. The members started to design the wiki.

## 08/10/2021

Aspects that make the project innovative were thought about. Medal criteria were looked over to avoid missing any.

## 08/17/2021

The PIs advised on how to improve certain protocols. The improvement of a part was started. The responsibility of writing texts for the wiki was split up. A moderator for the panel discussion was organized.

## 08/31/2021

The panel discussion was organized. A wiki party took place to write texts. Also, final things were planned for the panel discussion.

## 09/07/2021

A prototype of the wiki was designed. The lab books were revised. The title and abstract were written to be published on the iGEM site.

## 09/14/2021

The postcards were shipped to all participating teams. The presenters for the final presentation of our project were decided on. The escape room collaboration was finished.

## 09/21/2021

A final team roster was looked over and more wiki articles were finished.

## 09/28/2021

The safety form was finalized.

## 10/05/2021

Deadlines for the next few weeks were set and preparations for the wiki freeze were made.

## 10/12/2021

Final updates and revision of wiki texts.

* Team

  [Team](https://2021.igem.org/Team%3ADuesseldorf/Team)[Attributions](https://2021.igem.org/Team%3ADuesseldorf/Attributions)[Collaborations](https://2021.igem.org/Team%3ADuesseldorf/Collaborations)[Memes](https://2021.igem.org/Team%3ADuesseldorf/Memes)
* Project

  [Contribution](https://2021.igem.org/Team%3ADuesseldorf/Contribution)[Description](https://2021.igem.org/Team%3ADuesseldorf/Description)[Implementation](https://2021.igem.org/Team%3ADuesseldorf/Implementation)[Proof Of Concept](https://2021.igem.org/Team%3ADuesseldorf/Proof_Of_Concept)[Design](https://2021.igem.org/Team%3ADuesseldorf/Design)[Engineering](https://2021.igem.org/Team%3ADuesseldorf/Engineering)[Experiments](https://2021.igem.org/Team%3ADuesseldorf/Experiments)[Notebook](https://2021.igem.org/Team%3ADuesseldorf/Notebook)[Results](https://2021.igem.org/Team%3ADuesseldorf/Results)
* Parts

  [Parts](https://2021.igem.org/Team%3ADuesseldorf/Parts)[Improve](https://2021.igem.org/Team%3ADuesseldorf/Improve)
* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* Safety
  [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Duesseldorf/Engineering

Engineering | iGEM Team DD

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* Parts

  [Parts](https://2021.igem.org/Team%3ADuesseldorf/Parts)[Improve](https://2021.igem.org/Team%3ADuesseldorf/Improve)
* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

![background_image](https://static.igem.org/mediawiki/2021/6/61/T--Duesseldorf--img--eng-title.jpg)

# Engineering

---

##### Contents

## Gold-nanoparticle aptamer conjugates

### *1st iteration*

**Design:** In the beginning we had no experience in working with Lateral-Flow-Assays(LFAs). We oriented ourselves on the papers of Shijia Wu et al.[1](#citation1) and Guodong Liu et al. [2](#citation2) to get an idea of what we needed to do and on how to design and build our test.

**Build:** After getting everything we needed, it was time to start the first experiment. We started with the preparation of the gold nanoparticle (AuNPs)-aptamer-conjugates and the verification of the thiol-gold binding with a gel electrophoresis.

**Test:** In this experiment we already faced the first problem: the gold nanoparticle-solution turned from red to blue, which indicates that they have aggregated. When gold nanoparticles aggregate this is irreversible and they become unusable for our application.

**Learn:** After some research we found two possible causes for this aggregation. First, the NaCl or its specific concentration in the PBS buffer could have caused the AuNPs to aggregate. We learned that using a buffer with high ionic strength, like PBS, with citrate stabilized gold nanoparticles, can lead to aggregation. The aggregation occurs due to shield charging of the nanoparticles[3](#citation3). Second, the protocol itself could have some issues that we are not aware of. They synthesized their gold nanoparticles themselves, which might make a difference. Therefore we first needed to figure out what went wrong.

### *2nd iteration*

**Design:** To get to the bottom of which of the problems was the most likely one, we first wanted to figure out whether the buffer was causing the aggregation. And if it was the cause we wanted to know if it caused aggregation due to the NaCl concentration or something else in it.

**Build:** For that we prepared five different buffers with different concentrations of NaCl, PBS and sodium phosphate buffer, which does not contain NaCl.

**Test:** When we resuspended the AuNP-aptamer-solution in the five different probes, all five turned blue once again and were no longer usable for us.

**Learn:** This result showed us that the solutions we came up with did not fix our problem. We therefore came to the conclusion that if the aggregation was caused by the buffer, it was not related to the concentration.

### *3rd iteration*

**Design:** We then thought that maybe the citrate buffer our gold nanoparticles were delivered in might make them less stable. In order to test this hypothesis we tried our protocol again but this time we rebuffered the AuNPs before use.

**Build:** Four samples were set up. We washed the AuNPs and resuspended them in 1) milliQ water, 2) PB (phosphate buffer) and 3) PBS. Sample 4) was a control sample where the AuNPs stayed in the citrate buffer they came in. Additionally, we made two more samples and slowly added only 0.1 mM of PBS at a time until we reached our needed PBS concentration. This was done to test if adding the buffer more slowly, giving the AuNPs more time to adapt, could prevent them from aggregating

**Test:** When we rebuffered the AuNPs in different buffers, we had our first successful results. Only the sample that got resuspended in PBS turned blue. The others 2.- 4. stayed red.

![from top to bottom: PBS, PB, H2O, citrate](https://static.igem.org/mediawiki/2021/d/dd/T--Duesseldorf--img--eng-1.png)

Figure 1: from top to bottom: PBS, PB, H2O, citrate

In the additional experiment, in every step of adding small amounts of PBS, the AuNPs stayed red until the last time, when we added 10 mM PBS.

**Learn:** We concluded from this experiment that changing the buffer of the AuNPs was possible but PBS was not suitable for this. In the second experiment we could see that the pace and concentration at which the PBS is added can affect whether the AuNPs aggregate or not.

### *4th iteration*

**Design:** To test, if the newly generated samples stayed red during the further process, we used the PB and the citrate buffered sample from the previous experiment. We then tested if the AuNps could form conjugates with the aptamers. This was done via gel electrophoresis. Due to the red color of the AuNps no additional dyes are needed. If conjugates were formed we expected the samples to run more slowly than the control AuNPs, as they effectively increase in size.

**Build:** The further processing included an incubation step in which the aptamers should bind to the AuNps and two washing steps at the end of which the AuNps were resuspended in water. To see if there truly was conjugation between the AuNps and the aptamers, a 2% agarose gel was prepared in 0.5xTBE and run at 100 V for 90 min. 50% sucrose was added to each sample to increase their density so they would sink into the gel's pockets.

**Test:** The originally phosphate buffered sample turned blue during the washing steps. The originally citrate buffered sample and a control sample with AuNPs ran through the gel. Surprisingly, they passed through the gel very slowly, but at the same speed.

![1. lane: originally phosphate buffered AuNP-aptamer-conjugates; 2. lane: originally citrate buffered AuNP-aptamer-conjugates; 3. lane: control AuNPs](https://static.igem.org/mediawiki/2021/7/74/T--Duesseldorf--img--eng-2.png)

Figure 2: 1. lane: originally phosphate buffered AuNP-aptamer-conjugates; 2. lane: originally citrate buffered AuNP-aptamer-conjugates; 3. lane: control AuNPs

**Learn:** These results could either mean that there was no conjugation in the citrate buffered sample or the gel was too dense to separate the sample and the control.

### *5th iteration - success*

**Design:** Due to the inconclusive results from the prior experiment, we decided to try out a different agarose gel, this time with 1% agarose, and to use the originally citrate buffered AuNPs-aptamer-conjugates.

**Build:** The 1% agarose gel was placed in 0,5xTBE and run at 80 V for 90 min.

**Test:** Finally, the band of the AuNP-conjugates ran slower than the control AuNPs, which indicates that the conjugates have formed.

![1. lane: originally citrate buffered AuNP-aptamer-conjugates; 2. lane: control AuNPs](https://static.igem.org/mediawiki/2021/e/ee/T--Duesseldorf--img--eng-3.png)

Figure 3: 1. lane: originally citrate buffered AuNP-aptamer-conjugates; 2. lane: control AuNPs

**Learn:** From this experiment we learned that in order for the gold nanoparticles to not aggregate and bind to the aptamers, we could not change their buffer to anything but water. Furthermore, from now on we will exclude PBS and the included NaCl from our experiments with AuNPs, since this always leads to the aggregation of the AuNPs.

*Side note: Unfortunately, we were not able to recreate this again, so we had to walk through another Engineering cycle.*

### *6th iteration*

**Design:** Having researched our problem one more time, we found another protocol that stated that heating the aptamers could help[4](#citation4).

**Build:** 100 µM aptamers were heated to 90 °C and were allowed to cool down slowly to room temperature. 99 µl of these aptamers were mixed with a spatula tip SDS reacted for 30 min. at room temperature. 60 µl AuNPs and 10 µl aptamer-SDS-mix were combined and left to stand for 16 h at room temperature.

**Test:** The new AuNP-aptamer conjugates stayed red again and we ran them again on a 1% agarose gel to check if the conjugation worked. Unexpectedly the new conjugates ran faster than the control AuNP.

![left: new AuNPs-aptamer conjugates; right: control AuNPs](https://static.igem.org/mediawiki/2021/1/1c/T--Duesseldorf--img--eng-4.png)

Figure 4: left: new AuNPs-aptamer conjugates; right: control AuNPs

**Learn:** We doubt that the conjugation worked properly, since in all the papers we read, it is stated that the conjugates should run slower. We suppose that maybe due to the bound DNA, the AuNP-aptamer-conjugates have more negative charge than the unmodified AuNPs and therefore run faster than expected.

## References

1. Wu, S., Liu, L., Duan, N., Li, Q., Zhou, Y., & Wang, Z. (2018).

   Aptamer-Based Lateral Flow Test Strip for Rapid Detection of Zearalenone in Corn Samples.

   Journal of Agricultural and Food Chemistry 66(8), 1949-1954.

   [CrossRef](https://doi.org/10.1021/acs.jafc.7b05326)[Google Scholar](https://scholar.google.com/scholar?q=Aptamer-Based Lateral Flow Test Strip for Rapid Detection of Zearalenone in Corn Samples.)[Back to text](#intext1)
2. Liu, G., Gurung, A., & Qiu, W. (2019).

   Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin.

   Molecules 24(4), 756.

   [CrossRef](https://doi.org/10.3390/molecules24040756)[Google Scholar](https://scholar.google.com/scholar?q=Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin.)[Back to text](#intext2)
3. Ask an Expert: Why do gold nanoparticles aggregate? How do I prevent them from aggregating? What do I do if I see aggregation?. Nanohybrids.

   (n.d.). Retrieved on October 19, 2021. from <https://nanohybrids.net/pages/understanding-gold-nanoparticle-aggregation>

   [Back to text](#intext3)
4. Peng, Y., Li, L., Mu, X., & Guo, L. (2013).

   Aptamer-gold nanoparticle-based colorimetric assay for the sensitive detection of thrombin.

   Sensors and Actuators B: Chemical 177, 818-825.

   [CrossRef](https://doi.org/10.1016/j.snb.2012.12.004)[Google Scholar](https://scholar.google.com/scholar?q=Aptamer-gold nanoparticle-based colorimetric assay for the sensitive detection of thrombin.)[Back to text](#intext4)

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* Parts

  [Parts](https://2021.igem.org/Team%3ADuesseldorf/Parts)[Improve](https://2021.igem.org/Team%3ADuesseldorf/Improve)
* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* Safety
  [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Duesseldorf/Experiments

Experiments | iGEM Team DD

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* Parts

  [Parts](https://2021.igem.org/Team%3ADuesseldorf/Parts)[Improve](https://2021.igem.org/Team%3ADuesseldorf/Improve)
* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

![background_image](https://static.igem.org/mediawiki/2021/e/e7/T--Duesseldorf--img--experiment.png)

# Experiments

---

##### Contents

## Protocols

1. [Ampelpflanze FILESIZE: 40MB](https://static.igem.org/mediawiki/2021/c/c0/T--Duesseldorf--Lab_book_Ampelpflanze.pdf)
2. [SELEX FILESIZE: 21MB](https://static.igem.org/mediawiki/2021/0/09/T--Duesseldorf--Lab_book_SELEX.pdf)
3. [Proteam FILESIZE: 14MB](https://static.igem.org/mediawiki/2021/0/03/T--Duesseldorf--Lab_book_Proteam.pdf)
4. [Cell-SELEX FILESIZE: 3MB](https://static.igem.org/mediawiki/2021/e/ec/T--Duesseldorf--Lab_book_Cell-SELEX.pdf)
5. [Part-Improvement FILESIZE: 0.3MB](https://static.igem.org/mediawiki/2021/1/17/T--Duesseldorf--Lab_book_Part-Improvement.pdf)

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* Parts

  [Parts](https://2021.igem.org/Team%3ADuesseldorf/Parts)[Improve](https://2021.igem.org/Team%3ADuesseldorf/Improve)
* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* Safety
  [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

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# Team:Duesseldorf/Results

Results | iGEM Team DD

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* Team

  [Team](https://2021.igem.org/Team%3ADuesseldorf/Team)[Attributions](https://2021.igem.org/Team%3ADuesseldorf/Attributions)[Collaborations](https://2021.igem.org/Team%3ADuesseldorf/Collaborations)[Memes](https://2021.igem.org/Team%3ADuesseldorf/Memes)
* Project

  [Contribution](https://2021.igem.org/Team%3ADuesseldorf/Contribution)[Description](https://2021.igem.org/Team%3ADuesseldorf/Description)[Implementation](https://2021.igem.org/Team%3ADuesseldorf/Implementation)[Proof Of Concept](https://2021.igem.org/Team%3ADuesseldorf/Proof_Of_Concept)[Design](https://2021.igem.org/Team%3ADuesseldorf/Design)[Engineering](https://2021.igem.org/Team%3ADuesseldorf/Engineering)[Experiments](https://2021.igem.org/Team%3ADuesseldorf/Experiments)[Notebook](https://2021.igem.org/Team%3ADuesseldorf/Notebook)[Results](https://2021.igem.org/Team%3ADuesseldorf/Results)
* Parts

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* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)

![background_image](https://static.igem.org/mediawiki/2021/5/5a/T--Duesseldorf--img--ampelpflanze_%2818%29.jpeg)

# Results

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##### Contents

## Detection

**Goal**

Create a working sandwich Lateral-flow assay with our own aptamers for plant pathogens.

**Results**

We were able to prove that our preparation of the gold-nanoparticle(AuNP)-aptamer-conjugates was successful on one occasion. The successful conjugation of AuNPs with aptamers increases their effective size, reducing their running speed in the agarose gel. The control AuNPs run faster as their effective size is smaller.

![1. lane: originally citrate buffered AuNP-aptamer-conjugates; 2. lane: control AuNPs. ](https://static.igem.org/mediawiki/2021/5/5e/T--Duesseldorf--img--res-1.png)

Figure 1: 1. lane: originally citrate buffered AuNP-aptamer-conjugates; 2. lane: control AuNPs.

**Future directions**

Get devices like the AirJet dispenser, for easier dispensing of the control and test line, desiccators for a better drying process and the clamshell laminator to hold the single parts of the test in place, for an easier building process of the LFA.

## Ampelpflanze

**Goal**

A reporter plant that changes color according to the surrounding stressors.

**Results**

Transformation of *Arabidopsis thaliana* with the control construct induced a change of the coloration of the whole plant. This proved that the RUBY gene cassette was functional and capable of inducing the synthesis of betalain.

![Arabidopsis thaliana transformed with P35S_RUBY showing constitutive red coloring.](https://static.igem.org/mediawiki/2021/8/8c/T--Duesseldorf--img--res-2.jpg)

Figure 2: Arabidopsis thaliana transformed with P35S\_RUBY showing constitutive red coloring.

The seedlings grown from seeds of the transformed plants didn't exhibit their respective color.

**Future directions**

For the constitutively induced controls more transformations need to be done and more seedlings need to be screened. Additionally the stress inducible promoters still need to be extracted from gDNA and cloned in front of the reporter genes.

## Proteam

**Goal**

Express and purify one or more target proteins, so they can be used as targets for SELEX in the future

**Results**

We isolated one of our target proteins from cDNA, and were able to get it into the expression vector pET-21a(+) via Gibson assembly. The correct integration was also confirmed via [sequencing](https://static.igem.org/mediawiki/2021/2/20/T--Duesseldorf--UGT74F2_Sequencing.pdf)

![DNA-gel showing the amplification of UGT74F2 in the preceding PCR. The band can be observed at roughly 1400 bp, just as expected from UGT74F2. PR1 didn't show any amplification. The ladder used is a GeneRulerTM 1kb DNA ladder from Thermo ScientificTM.](https://static.igem.org/mediawiki/2021/4/48/T--Duesseldorf--img--res-3.jpg)

Figure 3: DNA-gel showing the amplification of UGT74F2 in the preceding PCR. The band can be observed at roughly 1400 bp, just as expected from UGT74F2. PR1 didn't show any amplification. The ladder used is a GeneRulerTM 1kb DNA ladder from Thermo ScientificTM.

We then successfully expressed said protein (UGT74F2) in *E. coli*.

![Coomassie-stained SDS gel of *E. coli* cell extract from before and after the induction with IPTG. After the induction there is a clear band at roughly 50 kDa, which is the size we expect UGT74F2 to have. The ladder is a PageRulerTM Plus Prestained protein ladder from ThermoScientificTM.](https://static.igem.org/mediawiki/2021/6/66/T--Duesseldorf--img--res-4.jpg)

Figure 4: Coomassie-stained SDS gel of \*E. coli\* cell extract from before and after the induction with IPTG. After the induction there is a clear band at roughly 50 kDa, which is the size we expect UGT74F2 to have. The ladder is a PageRulerTM Plus Prestained protein ladder from ThermoScientificTM.

**Future directions**

To actually use the protein as a target for SELEX it has to be purified first. As we added a His-tag to the protein, a His-tag-purification would be the way to go here.

## (Cell-)SELEX

**Goal**

Evolve aptamers for our test with the Cell-SELEX method.

**Results**

We were able to establish some parts of the Cell-SELEX process in our lab, for example the incubation of the library with the target cells and the digestion of double-stranded DNA into single stranded DNA. For confirmation, we used a 2 % TBE Agarose gel. If the digestion was successful, a different running speed through the gel would be expected. This can indeed be seen in the gel.

![Electrophoresis for digestion verification Lanes from left to right: 1. ssDNA (Clean up after digestion) 2. dsDNA Reference (Probe 1) 3. ssDNA (no clean up after digestion) 4. 50 bp ladder. ](https://static.igem.org/mediawiki/2021/b/b2/T--Duesseldorf--img--res-5.jpg)

Figure 5: Electrophoresis for digestion verification Lanes from left to right: 1. ssDNA (Clean up after digestion) 2. dsDNA Reference (Probe 1) 3. ssDNA (no clean up after digestion) 4. 50 bp ladder.

Even though the ladder did not separate properly, both single-stranded DNA (ssDNA) samples travelled significantly faster than the double-stranded DNA (dsDNA) reference.

Furthermore, we experimentally tested the optimal centrifugal forces for our *Pseudomonas syringae* cultures with the help of a cell-counter. First, we did a reference probe to get an idea of our baseline cell count. Next we centrifuged at different g-forces (150/500/1000/1500 x g) and took the supernatant to compare it with our reference. The fewer cells were counted, the more cells were pelleted. Another factor that we took into consideration is the "debris", cell counts that have a different diameter than our target cells. Only the peak was taken into consideration.

![The data generated by the Cell Counter. ](https://static.igem.org/mediawiki/2021/9/9d/T--Duesseldorf--img--res-6.png)

Figure 6: The data generated by the Cell Counter.

With the cell count / ml combined with the cell / debris ratio, we identified 1000 x g as the optimal g-force to minimize dead cells that can compromise our experiments while having sufficient pelletization.

![Cell Counter values](https://static.igem.org/mediawiki/2021/e/e8/T--Duesseldorf--img--res-7.png)

Figure 7: Cell Counter values

![Cell Count to debris ratio](https://static.igem.org/mediawiki/2021/2/26/T--Duesseldorf--img--res-8.png)

Figure 8: Cell Count to debris ratio

**Future directions**

Perform enough Cell-SELEX cycles, to evolve the aptamers that bind specifically to the cell surface of the plant pathogen *Pseudomonas syringae*.

* Team

  [Team](https://2021.igem.org/Team%3ADuesseldorf/Team)[Attributions](https://2021.igem.org/Team%3ADuesseldorf/Attributions)[Collaborations](https://2021.igem.org/Team%3ADuesseldorf/Collaborations)[Memes](https://2021.igem.org/Team%3ADuesseldorf/Memes)
* Project

  [Contribution](https://2021.igem.org/Team%3ADuesseldorf/Contribution)[Description](https://2021.igem.org/Team%3ADuesseldorf/Description)[Implementation](https://2021.igem.org/Team%3ADuesseldorf/Implementation)[Proof Of Concept](https://2021.igem.org/Team%3ADuesseldorf/Proof_Of_Concept)[Design](https://2021.igem.org/Team%3ADuesseldorf/Design)[Engineering](https://2021.igem.org/Team%3ADuesseldorf/Engineering)[Experiments](https://2021.igem.org/Team%3ADuesseldorf/Experiments)[Notebook](https://2021.igem.org/Team%3ADuesseldorf/Notebook)[Results](https://2021.igem.org/Team%3ADuesseldorf/Results)
* Parts

  [Parts](https://2021.igem.org/Team%3ADuesseldorf/Parts)[Improve](https://2021.igem.org/Team%3ADuesseldorf/Improve)
* Public Engagement

  [Communication](https://2021.igem.org/Team%3ADuesseldorf/Communication)[Human Practices](https://2021.igem.org/Team%3ADuesseldorf/Human_Practices)
* Safety
  [Safety](https://2021.igem.org/Team%3ADuesseldorf/Safety)