iGEM 2021 — Team TU-Eindhoven

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# Team:TU-Eindhoven/Notebook

[![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--TeamLogoWhite.png)](https://2021.igem.org/Team%3ATU-Eindhoven)

* Project

  [Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)
  [Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering)
  [Contribution](https://2021.igem.org/Team%3ATU-Eindhoven/Contribution)
* Lab

  [Experiments](https://2021.igem.org/Team%3ATU-Eindhoven/Experiments)
  [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook)
  [Parts](https://2021.igem.org/Team%3ATU-Eindhoven/Parts)
  [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept)
  [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results)
* [Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3ATU-Eindhoven/Implementation)
  [Communication](https://2021.igem.org/Team%3ATU-Eindhoven/Communication)
  [Education](https://2021.igem.org/Team%3ATU-Eindhoven/Education)
* [Safety](https://2021.igem.org/Team%3ATU-Eindhoven/Safety)
* Teams

  [Our Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)
  [Collaborations](https://2021.igem.org/Team%3ATU-Eindhoven/Collaborations)
  [Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)
  [Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)
  [Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)
* Awards

  [Medal requirements](https://2021.igem.org/Team%3ATU-Eindhoven/Medal_requirements)
  [Judging Form](https://igem.org/2021_Judging_Form?id=3972)
  [Awards](https://2021.igem.org/Team%3ATU-Eindhoven/Awards)

# Notebook

On this page, you can find short summaries of all the lab work we performed. You can click on the boxes for more information. In depth information about the results and achievements in the lab are described on the [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results) page and the [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept) page.

##### **Week 26 (June 28th - July 4th) - Preparing stock solutions**

We prepared the antibiotic stock solutions, the IPTG stock solutions and made LB medium. We accidentally made a solution of 25 mg/mL spectinomycin instead of 50 mg/mL, which means that we have to add double the amount of spectinomycin in the future (e.g. 200 µL instead of 100 µL for large cultures). Furthermore, we prepared medium for agar plates by autoclaving LB medium containing agar-agar.

##### **Week 27 (July 5th - July 11th) - ARG & TtrS/R (co-)transformation**

The ordered ARG, TtrS and TtrR plasmids were delivered in dry swaps in DH5 alpha cells and were used to make small cultures for Miniprep preparation. After overnight incubation, we isolated the plasmids from the DH5 alpha cells with the Qiagen Miniprep kit and co-transformed the TtrR and TtrS plasmids into *E. coli* BL21 (DE3) cells and transformed the ARG plasmid into different BL21 (DE3) cells.

Furthermore, we made small culture glycerol stocks from the small cultures and stored them in the -80 °C freezer. In addition, more agar plates were poured containing only kanamycin and plates with spectinomycin and chloramphenicol. Unfortunately, the co-transformation of TtrS/R did not work, since the Drigalski spatula was likely too hot during the spreading. Therefore we executed the co-transformation again. However, the second co-transformation did not result in colonies on the plates either. Therefore, we asked our supervisor Yan Ni for advice, and she recommended using SOC medium instead of LB medium during the incubation of the transformed cells. We repeated the co-transformation with the SOC medium and had colonies on the plates. The transformation with ARG did work the first time.

##### **Week 28 (July 12th - July 18th) - First troubles with ARG and TtrS/R**

This week we had planned to do the first ultrasound measurements, so we started with a small culture of ARG, made a large culture out of a prepared small culture and prepared the first phantoms containing the large cultures. During the measurement of the phantoms using the ultrasound, we could see a lot of white stripes on the bottom of the ultrasound images. Furthermore, we wanted to purify the ARG proteins to see if the cells did make the right proteins. However, due to a mistake in the protocol, we discarded our proteins and the protocol was not continued.

Additionally, we wanted to measure the GFP fluorescence of TtrS/R with different concentrations of inducers (to establish a dose-response) and therefore made a small culture and large culture. However, the LB medium that we used for the small cultures turned out to be contaminated and therefore no results were obtained. Furthermore, the tetrathionate inducer that we used should have been stored in the fridge. However, other researchers stored it at room temperature (for over a year), which likely resulted in the degradation of the tetrathionate. We noticed that it did not dissolve well enough in water and had a different color than was expected. Therefore, a new bottle of tetrathionate was ordered.

##### **Week 29 (July 19th - July 25th) - Improving ARG imaging**

Since the ultrasound images of last week were not clear and showed white stripes, we decided to discuss these results with an expert in ultrasound imaging: Hein de Hoop. We prepared phantoms with only PBS agar agar and one also mixed with LB medium. With his tips, we continued with a small culture, large culture and the ARG measurement. Since we only saved the pictures instead of the raw data, image processing was not possible. Therefore, we could not see the differences between the images before and after collapse. Furthermore, we purified the ARG proteins and measured their concentrations using the Nanodrop. More on the improvements we made in the ultrasound measurements with the help of Hein can be found on the [Engineering Succes](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering) page segment Ultrasound.

##### **Week 30 (July 26th - August 2nd) - Holiday**

Since most members of our team were on holiday, we did not perform lab work this week.

##### **Week 31 (August 3rd - August 8th) - TtrS/R GFP & mCherry measurements**

This week, we wanted to repeat the TtrS/R GFP measurement again with new ordered tetrathionate and new LB medium. Therefore, we did a co-transformation with TtrR and TtrS plasmids using SOC medium. Afterward, we did small culture and large culture and measured the GFP and mCherry fluorescence signals with the plate reader and the photospectrometer. The plate reader and the photospectrometer results showed the same fluorescence intensity for all samples and the blanco.

##### **Week 32 (August 9th - August 15th) - Site-directed mutagenesis for mutant ARG & wildtype ARG imaging**

Since our ARG plasmid was not Biobrick RFC[10] or RFC[1000] compatible, we had to insert 3 mutations in the DNA. Therefore, we did site-directed mutagenesis with our plasmid and made a small culture of the colonies for sequencing. Unfortunately, the sequencing results contained multiple non determined bases, which made the results not useful. Next to site-directed mutagenesis, we did a transformation and small culture of wildtype ARG and a large culture for ARG ultrasound imaging. During the ultrasound imaging, we received help from Hans-Martin Schwab, for more information on this see  [Engineering Succes](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering). He recommended using a different protocol, to reduce air bubbles, for making these phantoms by using a heating plate instead of the microwave for heating and mixing the agar agar with the PBS. We also used this protocol and made new ultrasound images. Furthermore, we purified the wildtype ARG proteins and made an SDS-PAGE. Since the SDS-PAGE was still too blue after shortly washing, we washed over the weekend and stained and washed again on Monday. Furthermore, the bands in the gel were not bright because the concentration of the proteins was too low.

##### **Week 33 (August 16th - August 22th) - Vary inducer concentrations for TtrS/R, amplify mutant ARG DNA and failed synthesis of design A plasmid**

This week, we made a picture of the SDS-page gel from last week and we repeated the TtrS/R GFP measurements, however, this time using different concentrations of dox, IPTG and tetrathionate, instead of a tetrathionate gradient. We made cultures and performed the GFP measurements again with the plate reader and the photospectrometer. The plate reader results gave similar fluorescent intensity for all samples and blanco. In contrast, when measuring the emission at different wavelengths at an excitation of 485 nm, the photospectrometer gave the expected peak with it’s maximum at the expected wavelengths. Furthermore, there a different fluorescence intensity was observed for the samples and the blank measurement and also different intensities between the samples were observed, as predicted. Therefore, further experiments were conducted using the photospectrometer.

We also amplified the amount of mutant ARG plasmid to obtain a higher concentration of mutant ARG for transformations. Lastly, we tried synthesising the design A plasmid, containing insert A, by restriction and ligation. However, we did not properly calculate the right amount of insert needed and therefore, the gel electrophoresis showed no bands and a smurred ladder. We choose not to continue with ligation and we first planned a meeting with expert Alexander Gräwe. More about the information we received from Alexander can be read on the [Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)) page.

##### **Week 34 (August 23th - August 29th) - Dox gradient for TtrS/R, successful restriction/ligation design A plasmid and first attempt for co-transformation of design A**

This week we measured the TtrS/R GFP intensity using a dox gradient. We performed the measurements for GFP and mCherry and the results are shown on the [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results) page. Furthermore, we successfully performed restriction and ligation of design A with insert A and tried to perform the co-transformation with the TtrS, TtrR and design A plasmids into BL21 (DE3) cells. Unfortunately, after co-transformation in BL21 (DE3) the agar plates showed no colonies. After meeting with Alexander Gräwe, it was concluded that the concentration of the design A plasmid was too low.

##### **Week 35 (July 30th - September 5th) - Different concentrations for wildtype ARG imaging, new attempts for co-transformation design A & dox concentration determination TtrS/R**

This week, we repeated wild type ARG ultrasound measurements, however, this time using a different gradient of IPTG concentrations than used in week 32. The wildtype ARG was purified again and placed on an SDS-PAGE gel and ultrasound measurements were performed. The ultrasound images were different from last time, which forced us to do some trouble shooting. Since the OD600 of our large cultures at the moment of induction was lower than the previous time (0.6 instead of 2), the phantoms contained not enough cells and thus the ultrasound images showed no difference between before and after collapse of the gas vesicles (except the concentration of 1000 µM IPTG). For the phantoms containing 10.000 µM IPTG, we suspect that the IPTG concentration was too high and the cells were killed, due to the toxic character of IPTG [1]. Moreover, the design A plasmid was amplified with a small culture and Miniprep was performed to obtain a higher concentration of plasmid. The previously executed co-transformation unfortunately did not work, so we repeated the experiment. However, this co-transformation also did not work, while the positive control showed colonies.

Besides, we performed TtrS/R GFP measurements again with a control and samples with 3 different dox concentrations with and without tetrathionate. The results can be seen on the [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results) page.

##### **Week 36 (September 6th - September 12th) - Contaminated dose-response of TtrS/R, TtrS/R competent cells for transformation of design A and preparation of part improvement**

Since we wanted to produce the full dose-response curve for the TtrS/R system, we repeated the very first experiment for TtrS/R, however, this time the optimum concentration of dox (0 ng/mL) was used. The thought behind this choice is elaborated on the [Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering) page. Unfortunately, we discovered that the LB medium used for the small cultures was contaminated. There was still some dose-response visible in the range as expected, but nothing could be concluded from these results, since all three controls failed.

Additionally, we tried the co-transformation of design A again by first making our TtrS/R cells competent. These cells already contain the TtrR and TtrS plasmids and the design A plasmid could be transformed into these competent cells. The co-transformation plates did not show any colonies. Furthermore, we started with preparations for part improvement by preparing agar plates and by transforming the wildtype pSB1C3-RFP coding device from the distribution kit. We made a small culture of the transformed cells and isolated the DNA using Miniprep.

##### **Week 37 (September 13th - September 19th) - Last attempt for the co-transformation design A & transfer to design B & restriction/ligation for part improvement**

This week, we tried the co-transformation of design A again with the TtrS/R competent cells. Since this did not work, we transferred to our design B approach and did the restriction and ligation of the design B plasmid. After ligation, we transformed the design B plasmid into Novablue cells. Unfortunately, this did not work so we tried the transformation again using XL10-Gold and NovaBlue cells. However, we still did not see any colonies on our plates. In addition, we tried the transformation of our mutant ARG again in BL21 (DE3) cells, which did not work.

Furthermore, for part improvement, we amplified the ordered 5’UTR insert using PCR and purified the DNA. Afterward, we restricted and ligated the pSB1C3 plasmid from the iGEM distribution kit with our 5’UTR insert and transformed them into BL21 (DE3) and DH5 alpha cells. We also transformed the wildtype pSB1C3-RFP coding device into BL21 (DE3). Since only colonies of the DH5 alpha cells were visible on the plates, we made a small culture of them and isolated the DNA with Miniprep. Furthermore, we transformed the wildtype pSB1C3-RFP coding device again in BL21 (DE3) and also the isolated ligated pSB1C3 in BL21 (DE3).

##### **Week 38 (September 20th - September 26th) - Transformation pSB1C3-5’UTR & sequencing**

This week, we continued with part improvement by amplifying the ligation product and the wildtype plasmid and continued with isolating the DNA using Miniprep. These plasmids were transformed into BL21 (DE3) cells, however, even after a few attempts, there were no colonies present on the agar plates, while the positive control was overgrown. To check the ligation product and the wildtype pSB1C3-RFP coding device, we sent these plasmids for sequencing. Furthermore, the restricted wildtype ARG and PCR product of the insert from design B were also sent for sequencing to check if the restriction sites were cut and if the PCR reaction worked.

##### **Week 39 (September 27th - October 3rd) - Transformation and amplification of pSB1C3 & synthesis of design B plasmid & dose-response of TtrS/R**

Since the previous dose-response curve attempts failed, we repeated this experiment again with the final concentrations of inducers and a tetrathionate gradient, as can be seen in the [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results). Alongside this experiment, we tried the restriction and ligation of design B again and transformed the ligation product into XL10-Gold and TOP10 cells. To achieve this, the insert B first had to be amplified by PCR. For part improvement, we amplified the 5’UTR insert with PCR and transformed the pSB1C3-RFP coding device plasmid into DH5 alpha and TOP10 cells.

##### **Week 40 (October 4th - October 10th) - Finally working transformations, sequencing and the last measurements**

This week, we repeated the restriction/ligation of design B plasmid for the last time, with newly prepared insert and plasmid, and transformed the ligation product into XL10-Gold and TOP10 cells. Since colonies appeared on all plates except the negative control, we made a small culture of the TOP10 and XL10-Gold colonies with the ligation product and isolated the DNA. We co-transformed the ligation product together with TtrS in XL10-Gold, TOP10 and BL21(DE3) cells which resulted in multiple plates with colonies and thus successfully transformed design B. Furthermore, we tried to co-transform design A plasmid again in BL21 (DE3) cells, which as expected did not grow.

In addition, we tried the restriction/ligation again for pSB1C3-5'UTR and successfully transformed the ligation product into TOP10 and DH5 alpha cells. We made a small culture and large culture, and measured the RFP fluorescence.

However, the laboratory we worked in suffered from a cleaning problem, which means that our large cultures were probably contaminated and therefore, the large cultures made this week have no reliable results. The ultrasound images had a lower intensity compared to the previous images, which caused the filters to fail. Therefore, a different filter was used, however, this filter influenced the results. Furthermore, no significant difference between induced and not induced images could be seen. The same problem can be seen with the RFP fluorescence measurements. Therefore, these results will be excluded.
Lastly, we sent our ARG insert A and B together with pSB1C3-5'UTR for sequencing.

##### **References**

1. Dvorak P, Chrast L, Nikel P, Fedr R, Soucek K, Sedlackova M et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway. Microbial Cell Factories [Internet]. 2015;14(1).

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

# Team:TU-Eindhoven/Engineering

[![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--TeamLogoWhite.png)](https://2021.igem.org/Team%3ATU-Eindhoven)

* Project

  [Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)
  [Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering)
  [Contribution](https://2021.igem.org/Team%3ATU-Eindhoven/Contribution)
* Lab

  [Experiments](https://2021.igem.org/Team%3ATU-Eindhoven/Experiments)
  [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook)
  [Parts](https://2021.igem.org/Team%3ATU-Eindhoven/Parts)
  [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept)
  [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results)
* [Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3ATU-Eindhoven/Implementation)
  [Communication](https://2021.igem.org/Team%3ATU-Eindhoven/Communication)
  [Education](https://2021.igem.org/Team%3ATU-Eindhoven/Education)
* [Safety](https://2021.igem.org/Team%3ATU-Eindhoven/Safety)
* Teams

  [Our Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)
  [Collaborations](https://2021.igem.org/Team%3ATU-Eindhoven/Collaborations)
  [Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)
  [Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)
  [Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)
* Awards

  [Medal requirements](https://2021.igem.org/Team%3ATU-Eindhoven/Medal_requirements)
  [Judging Form](https://igem.org/2021_Judging_Form?id=3972)
  [Awards](https://2021.igem.org/Team%3ATU-Eindhoven/Awards)

# Engineering Success

*“The key to success is to focus on goals, not obstacles”*

As with any other iGEM team, we ran into several challenges during the development of our project. These problems were solved by following the design cycle: Design, Build, Test, and Learn. As a result, we were able to achieve success by developing bacteria that included our biological system, building a kinetic model, developing new parts, and contributing to existing parts from the iGEM registry. Below, we will elaborate in more detail on three components of our project where we achieved success by implementing the design cycle.

Doxycycline
Ultrasound
Model

## Doxycycline

![](https://static.igem.org/mediawiki/2021/7/70/T--TU-Eindhoven--DesignBuild.png)

## Design

The sensing part of our project exists out of the two-component [TtrS/R sensing system](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept). To have more control over the expression of the TtrR and TtrS proteins, we wanted to make use of chemically controlled promoters. In the literature, we discovered that the pTac and the pLtetO-1 have been used by researchers [1]. These promoters are induced by IPTG and anhydrotetracycline (tetracycline derivative), and produce TtrS and TtrR respectively. Since plasmids including these promoters have been used in previous studies and were commercially available from *Addgene*, these plasmids have been used in our project.

To reproduce the results from the research paper as described in [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results), we had to determine the concentrations of all the inducers required for the test. Our supervisors advised us to make use of a university standard IPTG concentration of 0.1 mM and a series of tetrathionate (Ttr) concentrations, which is in line with the research paper (0.1 - 1000 µM). For the last inducer, anhydrotetracycline, it was more difficult to determine the optimal concentration, since this compound was not in stock at our university. Nevertheless, we found that doxycycline (dox), another tetracycline derivative, was present. Our supervisors advised us to search for a concentration often used and apply that concentration to obtain a full dose-response curve. We found an article [2] that uses a concentration of 250 ng/mL dox to induce the pLtetO-1 promoter inside BL21 (DE3) cells.

## Build

The building of the sensing part was relatively easy. We ordered the required plasmids from *Addgene* (pKD227 and pKD233.7-3), purified the plasmids, and co-transformed them into BL21 (DE3) cells. The initial co-transformation failed, however, the second one succeeded, giving us altered bacteria which should behave as the sensing part. To test this, we prepared all of the remedies as well as any additional materials we would need.

![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--DesignDesign.png)

![](https://static.igem.org/mediawiki/2021/5/5b/T--TU-Eindhoven--DesignTest.png)

## Test

To test the system, we made eight different cultures. The cultures consisted of five samples induced with different concentrations of tetrathionate, one negative control without tetrathionate induction, and two time-dependent samples. These time-dependent samples were induced with IPTG and dox at the same time as the rest of the samples, however, Ttr was added later. In addition to these samples, we also measured the emission of the buffer. The results showed that all eight samples emitted similar amounts of sfGFP, and the buffer emitted much less. Therefore, we concluded that the induction of the cells and the expression of sfGFP was successful and the right plasmids were present, however, the bacteria did not respond to Ttr as expected under these conditions.

## Learn

We went back to the aforementioned research paper [1] and checked the concentrations of inducers used in their experiments. It appeared that they used an IPTG concentration of 0.01 mM and anhydrotetracycline was not used at all for their sensing system, since the background expression was sufficient. Based on those statements, we composed an experiment making eight different samples altering the IPTG concentrations between 0.1 and 0.01 mM, the tetrathionate concentration between 0 and 1000 µM, and the dox concentration between 0 and 250 ng/mL.

![](https://static.igem.org/mediawiki/2021/c/c8/T--TU-Eindhoven--DesignLearn.png)

## Test and Learn Again

We put the aforementioned setup to the test, as can be read about in [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results). It was concluded that samples induced with 250 ng/mL dox, with and without tetrathionate, produced similar quantities of sfGFP. The other samples, without dox, only expressed sfGFP when induced with 1000 µM Ttr and (nearly) none when no Ttr was present. From these results, we concluded that the dox concentration should be further analyzed and possibly adjusted. We set up two experiments testing several dox concentrations ranging from 0.1 ng/mL to 250 ng/mL and tested the efficiency of the sensing part of our project. From this experiment, it turned out that concentrations of 250 ng/mL and 100 ng/mL were indeed overexpressing TtrR and that there was no difference between samples induced by IPTG concentrations under 10 ng/mL. Therefore, the advisors suggested using no doxycycline for the final design of our project, as supported by the research paper.

##### **References**

1. Daeffler, K., Galley, J., Sheth, R., Ortiz‐Velez, L., Bibb, C., Shroyer, N., Britton, R. and Tabor, J., 2017. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, [online] 13(4), p.923.
2. Mazumder M, Brechun K, Kim Y, Hoffmann S, Chen Y, Keiski C et al. An Escherichia coli system for evolving improved light-controlled DNA-binding proteins. Protein Engineering Design and Selection [Internet]. 2015 [cited 3 July 2021];28(9):293-302. Available from: https://academic.oup.com/peds/article/28/9/293/1605339

## Ultrasound

![](https://static.igem.org/mediawiki/2021/7/70/T--TU-Eindhoven--DesignBuild.png)

## Design

In our project, we want to image the ARG gas vesicles with ultrasound. Therefore, we needed to know which ultrasound machine and probe to use, how to handle them, and which script to employ. We looked into two articles that described ARG and tried to find two scripts that could be utilized to make ultrasound images and to collapse the gas vesicles [1,2]. Unfortunately, the scripts that were used by these particular researchers were not available and they did not respond to our requests. This was no problem since our university contains an ultrasound department, and already possesses a lot of standard research scripts we could use.

During the [meeting with Hans-Martin Schwäb](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices), an ultrasound expert at our university, we discovered that the article supplied a lot of detailed information about the settings and the setup of the ultrasound script and equipment. He told us to use a vertical sweep of the phantom, containing gas vesicle producing bacteria, with an L22-14V Verasonics probe. Some details about the research paper are further explained in [Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices) segment the Science.

## Build

After researching the paper and talking to ultrasound experts, we started with the building phase. During a new [meeting with Hein de Hoop](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices), another expert at our university, we got familiar with the ultrasound script and we were able to change most of the settings of the Verasonics standard ultrasound script to match our desires. To test this  [script](https://github.com/twarmink/iGEM-TU_Eindhoven.git), we imaged a standard phantom from the PULS/e lab at the university, adapting these settings to see if the settings work. In addition, the setup was constructed by clamping the transducer above the plate and making good contact between the probe and the phantom using a Phosphate-Buffered Saline (PBS) buffer.

![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--DesignDesign.png)

![](https://static.igem.org/mediawiki/2021/5/5b/T--TU-Eindhoven--DesignTest.png)

## Test

The next day, we tested the ultrasound setup and the script on our own samples, which were all induced with different IPTG concentrations and therefore, contained different concentrations of ARG vesicles. A clear transition between the liquid PBS buffer and the solid phantom was visible, however, a lot of background signal was visible as well (Figure 1). Furthermore, it was not clear to us whether we saw the PBS, the gas vesicles, or something else. So we went back to Hein de Hoop to help us out.

## Learn

After explaining our problem to Hein, he suggested clamping the transducer at an angle above the plate. The white stripes we saw on the images were probably created by the reflections of the sound waves at the bottom of the phantom. When angling the transducer, the reflections on the flat bottom of the phantom will bounce away from the transducer. Since the gas vesicles are round/ellipse shaped, the ultrasound signals hit the gas vesicles perpendicular, reflecting the signal back to the transducer. The noise reflections of the table will therefore not be visible on the images, while the vesicles will be visible.

![](https://static.igem.org/mediawiki/2021/c/c8/T--TU-Eindhoven--DesignLearn.png)

Accordingly, we changed the setup of the ultrasound and tested new phantoms with the vertical transducer and by angling the transducer. This time, Hans-Martin Schwäb joined us in the lab to check the results and to help us configure some minor settings, which had to be set manually each time. The angled images indeed showed better [results](https://2021.igem.org/Team%3ATU-Eindhoven/Results), than the vertical images. During this measurement session, Hans-Martin also explained a little bit about post-image processing and he pointed out that we should save the raw data instead of the images themselves. Not only is the quality of raw data higher than the quality of the images, image processing is also easier with raw data. We implemented this right away.

We had another meeting with Hans-Martin to design a script that could read the data and could calculate the difference between the images before and after the collapse of the gas vesicles (using the in-phase Quadrature, IQ). That way we could remove all background signals (e.g. from the agar) and it should be able to visualize only the gas vesicles. All latter ultrasound measurements have been performed with this script, this processing, and this setup. For all results please look at [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results) segment ARG1 reporter system.

![](https://static.igem.org/mediawiki/2021/0/0f/T--TU-Eindhoven--EngineeringSuccesUltrasoundFig1.png)

Figure 1: The difference in before and after collapse images, imaged with ultrasound with on the left with a straight probe and on the right with an angled probe.

##### **References**

1. Bourdeau, R., Lee-Gosselin, A., Lakshmanan, A. *et al.* (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. *Nature* 553, 86–90. <https://doi.org/10.1038/nature25021>
2. Lakshmanan, A., Lu, G., Farhadi, A. *et al.* (2017) Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI. *Nat Protoc* 12, 2050–2080. <https://doi.org/10.1038/nprot.2017.081>

## Model

During the development of our kinetic model, we ran through several iterations of the design cycle. Our supervisor, Tom de Greef, supported us during this process. Tom de Greef is an Associate Professor of Synthetic Biology in the Department of Biomedical Engineering and specialized in the modeling and simulation of biomolecular networks. Tom gave us the advice to use the Matlab add-on SimBiology and pointed out that it was important to keep the design cycle in mind during the development of a computational model. So instead of trying to build the entire model in one step, it is important to make intermediate, simplified models. These simplified models can be tested and validated before expansion. In this way, we were able to correct our mistakes and learn from them before moving on to a more complicated model.

![](https://static.igem.org/mediawiki/2021/7/70/T--TU-Eindhoven--DesignBuild.png)

## Design

After deciding on our concept in the early stages of our project, we determined what type of model we wanted to develop. We researched previous iGEM teams and found that the most useful model for our project would be a kinetic model of the TtrS/R two-component system. We set up a meeting with prof.dr.ir. Tom de Greef to discuss the main components of our model and how we want to achieve these. We established that our output should consist of a dose-response curve, from which we can derive how much GFP can be obtained for different amounts of tetrathionate. Later, this model can be expanded to derive the number of gas vesicles that are produced.

We started by searching the literature for the chemical reactions that take place in the transcription and translation of our two-component system, and the signaling cascade induced by tetrathionate. Various versions were made and in each version, more chemical reactions were added, thus expanding the model to be more extensive and more realistic. We decided on building the following five versions, each containing the components from the previous version and an additional extension.

* A model consisting of only the two-component sensing pathway, and the activation of GFP. This version contains a constant concentration of TtrS and TtrR, no delaying steps, and no degradations are taken into account.
* Expanded with the transcription and translation of TtrS and TtrR under the same promoter. The activation of TtrS/R transcription is modeled in one step, and no delaying steps or degradations are taken into account in this version.
* Expanded with a more elaborate activation of TtrS/R transcription modeled in multiple steps.
* Taking into account delaying steps and degradation reactions.
* Including the activation of acoustic reporter genes and the formation of gas vesicles.

After the chemical reactions were established, we had to search for reaction rates. We did this by looking at similar models and searching for experimental data in the literature. In addition, initial concentrations had to be determined for some of the species. Some of these concentrations could be retrieved from the lab protocols, such as the doses of tetrathionate and IPTG that are added. Other concentrations had to be calculated using values found in literature, such as the number of ribosomes or RNA polymerase per E. coli cell. After the reactions, rates, and initial concentrations were determined, we could start building the model.

## Build

As mentioned before, the program that was used to build our kinetic model is the MATLAB add-on SimBiology. For this program, it is not necessary to calculate the ordinary differential equations manually, as this is all done automatically. In this program, we entered the chemical reactions, rates, and concentrations that were previously determined. Naturally, we only entered the values necessary for the reactions that we were modeling in that particular version. A more in-depth description of our method can be found on [Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model), segment method.

![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--DesignDesign.png)

![](https://static.igem.org/mediawiki/2021/5/5b/T--TU-Eindhoven--DesignTest.png)

## Test

After building the models, we used the extra function of SimBiology called SimBiology Analyzer to simulate the model. With this program, you can simulate the expression of each species in the model over time. For each version, we analyzed several things to validate the accuracy of the model. Firstly, we checked whether the simulation only contained positive, logical values. We validated whether the protein and mRNA expression over time could be justified when looking at the chemical reactions that are happening in the TtrS/R system. In addition, we validated whether the total amount of TtrR and TtrS stayed stable, by summing the concentrations of all proteins either bound or unbound. The total concentration TtrR and TtrS should show a linear increase over time at first, and eventually stabilize. Further, the effect of the degradation reactions was checked by comparing the versions including degradation with the versions excluding degradation.

## Learn

Testing of the versions often resulted in errors or inaccuracies which had to be resolved in order to expand the model with extra reactions. When validation failed, the first step was to trace and analyze the error. Errors could often be resolved by adjusting the simulation settings. Inaccuracies in the models could have different origins, such as inaccurate rate constants, initial concentrations, or reactions. Thus, when unrealistic protein or mRNA expression was found in one of the simulations, our initial action response was to delve back into the literature to improve our input parameters. If we could not find the origin of the error, we contacted our supervisor for additional support. This way we were able to solve the problems that occurred in each version, before moving on to the next version.

![](https://static.igem.org/mediawiki/2021/c/c8/T--TU-Eindhoven--DesignLearn.png)

All results of this model can be found at [Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model), segment results. Right now, TtrS and TtrR transcription and translation are modeled equally. For future work, the model could be extended by including a more realistic transcription and translation of TtrS and TtrR on two different plasmids. Further, the parameters used in the model could be improved by conducting experimental measurements to determine the unknown reaction rates instead of estimating them. This way, the simulation of the system will become more accurate.

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[![](https://static.igem.org/mediawiki/2021/a/ae/T--TU-Eindhoven--LinkedInLogoWhite.png)](https://www.linkedin.com/company/igem-tu-eindhoven/mycompany/?viewAsMember=true)

###### IBDetection

Non-Invasive Monitoring of IBD using Genetically Engineered Bacteria as Living Sensors.

###### Project

[Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)

[Lab](https://2021.igem.org/Team%3ATU-Eindhoven/Results)

[Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)

[Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)

###### About Us

[Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)

[Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)

[Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)

[Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)

###### Contact

PO-box 513, 5600MB Eindhoven, Netherlands

igem@tue.nl

© 2021 Copyright:
[iGEM TU Eindhoven](https://2021.igem.org/Team%3ATU-Eindhoven)

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

# Team:TU-Eindhoven/Experiments

[![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--TeamLogoWhite.png)](https://2021.igem.org/Team%3ATU-Eindhoven)

* Project

  [Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)
  [Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering)
  [Contribution](https://2021.igem.org/Team%3ATU-Eindhoven/Contribution)
* Lab

  [Experiments](https://2021.igem.org/Team%3ATU-Eindhoven/Experiments)
  [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook)
  [Parts](https://2021.igem.org/Team%3ATU-Eindhoven/Parts)
  [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept)
  [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results)
* [Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3ATU-Eindhoven/Implementation)
  [Communication](https://2021.igem.org/Team%3ATU-Eindhoven/Communication)
  [Education](https://2021.igem.org/Team%3ATU-Eindhoven/Education)
* [Safety](https://2021.igem.org/Team%3ATU-Eindhoven/Safety)
* Teams

  [Our Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)
  [Collaborations](https://2021.igem.org/Team%3ATU-Eindhoven/Collaborations)
  [Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)
  [Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)
  [Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)
* Awards

  [Medal requirements](https://2021.igem.org/Team%3ATU-Eindhoven/Medal_requirements)
  [Judging Form](https://igem.org/2021_Judging_Form?id=3972)
  [Awards](https://2021.igem.org/Team%3ATU-Eindhoven/Awards)

# Experiments

*“When all else fails, read the instructions”*

Here, you can find all the protocols used in our project. More detailed information about the execution of our project can be found on  [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook). Our results can be found on the  [Improvement](https://2021.igem.org/Team%3ATU-Eindhoven/Improve),  [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results) and  [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept%20).

##### **Overview of all the Experiments**

All experiments executed during our project with their corresponding protocols can be found in the following booklet.

[IBDetection Experiments](https://static.igem.org/mediawiki/2021/5/52/T--TU-Eindhoven--ExperimentsOverAllBooklet.pdf%20)

##### **General Protocols**

The general protocols we used are listed below.

 [Co-transformation](https://static.igem.org/mediawiki/2021/7/74/T--TU-Eindhoven--ExperimentsCoTransformation.pdf)

 [Gel electrophoresis](https://static.igem.org/mediawiki/2021/0/05/T--TU-Eindhoven--ExperimentsGelElectrophoresis.pdf)

 [Gel extraction](https://static.igem.org/mediawiki/2021/b/bd/T--TU-Eindhoven--ExperimentsGelExtraction.pdf)

 [Large culture & protein Expression](https://static.igem.org/mediawiki/2021/2/21/T--TU-Eindhoven--ExperimentsLargeCultureProteinExpression.pdf)

 [Making competent cells](https://static.igem.org/mediawiki/2021/e/e9/T--TU-Eindhoven--ExperimentsMakingCompetentCells.pdf)

 [Making TSS solution](https://static.igem.org/mediawiki/2021/b/bb/T--TU-Eindhoven--ExperimentsMakingTSSSolution.pdf)

 [Medium & pouring agar plates](https://static.igem.org/mediawiki/2021/3/3a/T--TU-Eindhoven--ExperimentsMediumAgarPlates.pdf)

 [Multi Site-directed mutagenesis](https://static.igem.org/mediawiki/2021/9/9c/T--TU-Eindhoven--ExperimentsMultisitedirectedMutagenesis.pdf)

 [Nanodrop](https://static.igem.org/mediawiki/2021/8/8d/T--TU-Eindhoven--ExperimentsNanodrop.pdf)

 [PCR amplification & purification](https://static.igem.org/mediawiki/2021/6/6f/T--TU-Eindhoven--ExperimentsPCR.pdf)

 [Qiagen miniprep](https://static.igem.org/mediawiki/2021/5/54/T--TU-Eindhoven--ExperimentsQiagenMiniPrep.pdf)

 [Restriction & Ligation](https://static.igem.org/mediawiki/2021/0/03/T--TU-Eindhoven--ExperimentsRestrictionLigation.pdf)

 [Sequencing](https://static.igem.org/mediawiki/2021/1/19/T--TU-Eindhoven--ExperimentsSequencing.pdf)

 [Small culture](https://static.igem.org/mediawiki/2021/f/f4/T--TU-Eindhoven--ExperimentsSmallCulture.pdf)

 [Stock solutions](https://static.igem.org/mediawiki/2021/b/be/T--TU-Eindhoven--ExperimentsStockSolution.pdf)

##### **Two component TtrS/R protocols**

The specific protocol for the two component TtrS/R system is listed below.

 [GFP measuring](https://static.igem.org/mediawiki/2021/1/19/T--TU-Eindhoven--ExperimentsTtrSRProtocol.pdf)

##### **Acoustic Reporter Genes Protocols**

The specific protocols for the Acoustic reporter genes are listed below.

 [ARG Imaging](https://static.igem.org/mediawiki/2021/b/bc/T--TU-Eindhoven--ExperimentsARGImaging.pdf)

 [ARG Purifying](https://static.igem.org/mediawiki/2021/f/fd/T--TU-Eindhoven--ExperimentsARGPurifying.pdf)

 [SDS-PAGE](https://static.igem.org/mediawiki/2021/5/5c/T--TU-Eindhoven--ExperimentsARGSDSPage.pdf)

##### **Improvement**

The specific protocol for the part Improvement is listed below.

 [mRFP1 Fluorescence measurement](https://static.igem.org/mediawiki/2021/1/1f/T--TU-Eindhoven--ExperimentsImprovement.pdf)

[![](https://static.igem.org/mediawiki/2021/e/ed/T--TU-Eindhoven--FacebookLogoWhite.png)](https://www.facebook.com/iGEMTUEindhoven)
[![](https://static.igem.org/mediawiki/2021/b/b5/T--TU-Eindhoven--TwitterLogoWhite.png)](https://twitter.com/eindhoven_igem)
[![](https://static.igem.org/mediawiki/2021/8/86/T--TU-Eindhoven--InstagramLogoWhite.png)](https://www.instagram.com/igem_eindhoven/)
[![](https://static.igem.org/mediawiki/2021/a/ae/T--TU-Eindhoven--LinkedInLogoWhite.png)](https://www.linkedin.com/company/igem-tu-eindhoven/mycompany/?viewAsMember=true)

###### IBDetection

Non-Invasive Monitoring of IBD using Genetically Engineered Bacteria as Living Sensors.

###### Project

[Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)

[Lab](https://2021.igem.org/Team%3ATU-Eindhoven/Results)

[Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)

[Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)

###### About Us

[Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)

[Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)

[Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)

[Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)

###### Contact

PO-box 513, 5600MB Eindhoven, Netherlands

igem@tue.nl

© 2021 Copyright:
[iGEM TU Eindhoven](https://2021.igem.org/Team%3ATU-Eindhoven)

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

# Team:TU-Eindhoven/Results

[![](https://static.igem.org/mediawiki/2021/4/4e/T--TU-Eindhoven--TeamLogoWhite.png)](https://2021.igem.org/Team%3ATU-Eindhoven)

* Project

  [Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)
  [Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering)
  [Contribution](https://2021.igem.org/Team%3ATU-Eindhoven/Contribution)
* Lab

  [Experiments](https://2021.igem.org/Team%3ATU-Eindhoven/Experiments)
  [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook)
  [Parts](https://2021.igem.org/Team%3ATU-Eindhoven/Parts)
  [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept)
  [Results](https://2021.igem.org/Team%3ATU-Eindhoven/Results)
* [Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3ATU-Eindhoven/Implementation)
  [Communication](https://2021.igem.org/Team%3ATU-Eindhoven/Communication)
  [Education](https://2021.igem.org/Team%3ATU-Eindhoven/Education)
* [Safety](https://2021.igem.org/Team%3ATU-Eindhoven/Safety)
* Teams

  [Our Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)
  [Collaborations](https://2021.igem.org/Team%3ATU-Eindhoven/Collaborations)
  [Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)
  [Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)
  [Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)
* Awards

  [Medal requirements](https://2021.igem.org/Team%3ATU-Eindhoven/Medal_requirements)
  [Judging Form](https://igem.org/2021_Judging_Form?id=3972)
  [Awards](https://2021.igem.org/Team%3ATU-Eindhoven/Awards)

# Results

*“Once you see results, it becomes an addiction”*

[IBDetection](https://2021.igem.org/Team%3ATU-Eindhoven/Description) is a time-efficient, non-invasive, cost-effective, specific, and easy-to-use method of [monitoring IBD patients](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices). To prove that this approach is feasible, we designed a series of characterization experiments as explained on the [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept) page. The results for each of the three major characterizations (TtrS/R sensing system, ARG1 reporter system, and the entire sensor) will be discussed in this section.

## Summary

In short, we were able to confirm the sensitivity of the tetrathionate (Ttr) sensor in various experiments, resulting in a full dose-response curve for the TtrS/R sensing system, which detects Ttr and produces sfGFP as output (n=1). Besides, for the ARG1 reporter system, we were able to detect gas vesicles using ultrasound equipment and showed that we can increase protein production with the induction of a promoter. At last, we were able to create the engineered plasmids we designed. Unfortunately, due to time restrictions and troubles during the performance of the experiments, we had no time to test the plasmids. Nevertheless, based on the current results and the established model, we can envision a proper functioning of IBDetection in bacteria in relevant context.

## TtrS/R sensing system

As explained in our [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept), the TtrS/R sensing system is characterized by several experiments. The sensing system, consisting of purified pKD227 and pKD233.7-3 plasmids, was co-transformed into BL21 (DE3) cells (Figure 1a). The red color suggests the expression of the constitutive mCherry, a component of the pKD233.7-3 plasmid, confirming the successful transformation of the plasmid into the cells. The expression of mCherry was used to quickly determine which small cultures contained our plasmids (Figure 1b). Hereafter, large cultures were grown and used for the experimental measurements.

![](https://static.igem.org/mediawiki/2021/c/c7/T--TU-Eindhoven--ResultsFig1-1.png)

Figure 1: a) Agar plate with BL21 (DE3) containing the TtrR/S sensing plasmids. b) Small cultures of BL21 (DE3) in LB medium. Two cultures (left) do not contain the right cells and two cultures (right) contain the TtrS/R sensing system.

During the first sfGFP and mCherry measurements, results showed that the bacteria did not respond to varying Ttr concentrations (Figure 2). Since the co-transformations worked and the ordered plasmids had not been changed, we suspected the inducers to cause these unexpected results. Therefore, eight different combinations of Ttr, doxycycline (dox), and IPTG were tested for induction, by measuring sfGFP fluorescence. Through this experiment, it could be concluded that the sensor is working properly, however, the concentration of 250 ng/mL dox causes overexpression of the TtrR protein, resulting in high sfGFP expression (Figure 2b & c). To  [optimize the sensor](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering), several concentrations of dox were tested, and it was discovered that the concentration of dox equal to or smaller than 10 ng/mL gives rise to a tetrathionate sensor (Figure 2d & e). Based on these results and results from the literature, we choose to further test the sensor system without dox. In addition, to see whether the purification step in the protocol was necessary, we tested the emission inside *E. coli* BL21 (DE3) (Figure 2d) and in lysate suspended in PBS buffer (data not shown). We concluded that lysis of the bacteria was not necessary and therefore, was no longer performed. For more details about our progress, please read our [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook).

![](https://static.igem.org/mediawiki/2021/c/c9/T--TU-Eindhoven--ResultsFig2.png)

Figure 2: Normalized1 GFP emissions of the TtrS/R system induced with various concentrations doxycycline, IPTG and tetrathionate(Ttr). a) Dose-response curve measured in BL21 (DE3) lysate, induced with 0.1 mM IPTG, 250 ng/mL dox and different concentrations Ttr. b) Signal measured in BL21 (DE3) lysate, induced with 0.1 mM IPTG, with and without dox (250 ng/mL), and with and without Ttr (1000 µM). c) Signal measured in BL21 (DE3) lysate, induced with 0.01 mM IPTG, with and without dox (250 ng/mL), and with and without Ttr (1000 µM). d) Signal measured in BL21 (DE3), induced with 0.1 mM IPTG, 1000 µM Ttr and various concentrations dox. e) Signal measured in BL21 (DE3), induced with 0.1 mM IPTG, with and without 1000 µM Ttr and various concentrations dox.

With the optimal inducer concentrations for the bacteria to produce TtrR and TtrS, we performed one more characterization experiment with varying Ttr concentrations, which resulted in the dose-response curve visible in Figure 3.

![](https://static.igem.org/mediawiki/2021/b/b5/T--TU-Eindhoven--ResultsFig3.png)

Figure 3: Normalized1 GFP emissions of the TtrS/R system induced with various concentrations doxycycline, IPTG and tetrathionate.1 a) Dose-response curve measured in BL21 (DE3), induced with 0.1 mM IPTG, 0 ng/mL dox and different concentrations Ttr. In addition a positive control (0.1 mM IPTG, 250 ng/mL dox and 1000 µM Ttr) and a negative control (no IPTG, no dox, no Ttr) were added. b) The dose-response from figure 3a plotted in a line-plot with logarithmic scale (EC50 = 49.1 ± 2.3 µM, n = 1).

## Conclusion

Our results show a dose-response between 10 and 100 µM, and a EC50 of 49.1 ± 2.3 µM (n=1). The dose-response results correspond to the results of Daeffler et al. [1], who characterized the TtrS/R system and found a dose-response roughly between 10 and 100 µM Ttr with a EC50 of 50 ± 3 µM [1]. Hereby, we confirmed the reproduction of the sensing system of Daeffler et al. [1].

## ARG1 reporter system

We conducted various experiments to characterize the ARG1 reporter system, as explained in detail in the [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept). The reporter system was transformed into BL21 (DE3) cells using the purified pET28a T7-ARG1 plasmid DNA strand (Figure 4). A colony was picked and the cells were used to grow large cultures, which were induced with different concentrations of IPTG. These large cultures have been used to purify the proteins, which were analyzed using SDS-PAGE for the expression of gas vesicle proteins, and also used to create a phantom that can be analyzed using ultrasound equipment.

![](https://static.igem.org/mediawiki/2021/b/b4/T--TU-Eindhoven--ResultsFig4-1.png)

Figuur 4: Agar plate with BL21 (DE3) containing the ARG reporter plasmid.

The ultrasound measurement appeared to be more difficult to perform than anticipated as described in [Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering). After receiving advice from experts and editing of the  [scripts](https://github.com/twarmink/iGEM-TU_Eindhoven.git), we performed a successful ultrasound measurement in which a signal was acquired that corresponds to the gas vesicles. The signal showed a decreasing intensity, while decreasing the amount of IPTG concentration used for the induction (Figure 5). In addition to the ultrasound images, we purified the gas vesicles by centrifuging the lysed cells (in PBS buffer) and discarded the pellet. The resulting solution was used for an SDS-PAGE to check whether the gas vesicle proteins (all < 35 kDa) would be visible. A band around 30 kDa, indicating the proteins GvpN, GvpL, GvpT [2], supported the results from the ultrasound. Furthermore, there is a visible gradient, which corresponds to the decreasing concentration of (at least one of the) gas vesicle proteins (Figure 6).

![](https://static.igem.org/mediawiki/2021/2/24/T--TU-Eindhoven--ResultsFig5.png)

Figure 5: Ultrasound images of phantoms containing large culture with BL21 (DE3) induced with different concentrations IPTG. The images were made with the transducer at an angle with respect to the phantom containing 15 mL of 1% agar agar in PBS mixed with 15 mL large culture. Left) raw images before (pre) and after (post) running the collapse script (white signal is a reflection of the ultrasound signal). Right) processed difference between the pre and post collapse images. The white signal relates to the concentration gas vesicles.

![](https://static.igem.org/mediawiki/2021/d/d0/T--TU-Eindhoven--ResultsFig6.png)

Figure 6: SDS-PAGE of the centrifuge purified ARG proteins. On the left a Precision Plus ProteinTM All Blue Standards was used. Than there are three slots loaded with purified protein from wild type ARG large culture induced with 1000 µM, 10 µM and 0.1 µM IPTG respectively. Just as in the ultrasound a gradiënt is visible in intensity of a band around 30 kDa (red rectangle), suggesting successful control over the production of gas vesicle proteins.

The experiment was repeated two more times, though these results were not as expected. Both experiments failed due to different reasons resulting in ultrasound images without gas vesicles visible and with SDS-PAGE gels containing a lot of bands that might correspond to the gas vesicles, however, not showing any gradiënt. Therefore, these bands might also be just regular constitutive proteins from the bacteria which haven’t been purified during the process.

## Conclusion

Nevertheless, we have been able to visualize gas vesicles and the proteins by ultrasound and by SDS-PAGE. Additionally, we were able to show a gradiënt in the gas vesicle (protein) concentration by both methods, which outlines the potential of the use of ARG1 as an output system for IBDetection.

## Sensor

For the introduction of the tetrathionate sensor with pTtrB activated ARG1 (design A), and the tetrathionate sensor with transferred TtrR and pTtrB activated ARG1 (design B) into BL21 DE3), we first had to synthesize the plasmids that we explained in the [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept).
The plasmids were created by the restriction of the pET28a\_T7-ARG1 plasmid and were ligated with the required inserts, containing SgrAI and HF-NheI restriction sites. The restricted products were purified by gel electrophoresis followed by ligation.
Since the distances between two bands of restricted and unrestricted DNA were small, the gel extraction was difficult. Therefore, the restricted plasmid (negative control) was transformed parallel to the transformation of the ligation product. This was done, to check if the colonies are actually the ligated product or if the colonies contain unrestricted plasmid contamination. Fortunately we had serval successful and pure gel extractions resulting in proper ligation products. For more details check [Experiments](https://2021.igem.org/Team%3ATU-Eindhoven/Experiments) and [Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook).

## Results Design A

## Results Design B

For design A, we were able to successfully transform the pET28a\_pTtrB-ARG1 in XL10-Gold and purify the DNA. The next step was to introduce both (original) plasmids of the sensing system, containing the TtrS/R sensing system, together with this newly synthesized plasmid into a BL21 (DE3). After multiple failed attempts of introducing all the plasmids in a single co-transformation, we learned that it is also possible to do it in multiple steps. We made a culture of our BL21 (DE3) cells containing both plasmids for the TtrS/R sensing system and made them competent. We added the third plasmid required for design A to these competent cells, but this transformation failed as well. As described in the [Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices) segment the Science, we already considered this risk in the early stages of the project. Therefore, we were prepared to switch our research to testing design B and canceled experiments with design A, and all focus was set on design B.

For design B, we synthesized and transformed the pET28a\_TetR-TtrR-pTtrB185-269-ARG1 plasmid into XL10-Gold. After some failures, we repeated the restriction ligations and tried to transform the product into competent *E. coli* TOP10 and XL10-Gold cells. In the third attempt, colonies were grown on the plates, so it can be concluded that the required plasmid was successfully transformed. This plasmid was then amplified, purified and successfully co-transformed into BL21 (DE3), XL10-Gold, and TOP10 cells together with the p15a\_TtrS plasmid to create design B (Figure 7). This means that we were able to successfully create several types of bacterial cells, containing our engineered system.

![](https://static.igem.org/mediawiki/2021/9/9c/T--TU-Eindhoven--ResultsFig7.png)

Figure 7: Agar plates with bacteria containing both plasmids required for design B and thus IBDetection to work. a) BL21 (DE3) b) XL10-gold c) TOP10

## Conclusion

Due to the troubles encountered during the synthesis of both design A and design B, no time was left to actually characterize the co-transformed cells containing the IBDetection sensor-reporter system. Nevertheless, we are able to predict that the system should be successful. If you want to know why, take a look at the [Proof of Concept](https://2021.igem.org/Team%3ATU-Eindhoven/Proof_Of_Concept).

## Project Achievements

## Succes

As described above, we had several successes inside the lab.

* Reproduce and characterize the TtrS/R sensing system
* Create a full dose-response curve for the TtrS/R sensing system
* Reproduce the ARG1 reporter system
* Visualize gas vesicles with ultrasound and SDS-PAGE
* Create a plasmid with ARG1 gene under the pTtrB185-269 promoter.
* Create a plasmid with ARG1 gene under the pTtrB185-269 promoter with an additional TtrR and tetR protein upstream.
* Co-transform IBDetection (design B) in *BL21 (DE3), TOP10* and *XL10-Gold*.

## Road to Succes

To reach the above successes, we had to test and adapt regularly. The most important developments were:

* Doxycycline concentration optimization ([Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering))
* Ultrasound set-up optimization ([Engineering Success](https://2021.igem.org/Team%3ATU-Eindhoven/Engineering))
* Restriction & ligation adjustments ([Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices))
* Repeated transformations in various types of *E. coli* competent cells ([Notebook](https://2021.igem.org/Team%3ATU-Eindhoven/Notebook))

## Future Goals

Due to time constraints, several experiments have not yet been done and other experiments could only be conducted once. Therefore we first have to test the complete sensor and than check for the reproducibility of our results. This reproducibility is of great importance for clinical application. Therefore, it is necessary to repeat the characterization experiments that have been performed, to see if the same intensity and dose-response are measured every time. After confirming this, further research should be done towards the final design of our project, including tests with constitutive promoters for TtrR and TtrS, experiments in Lactic Acid Bacteria (e.g. *L. plantarum* WCFS-1), and determination of the number of cells required for the pill. Moreover, we should test the safety of the GMO, various types of possible pills and differences between users. Details about this can be read on the [Implementation](https://2021.igem.org/Team%3ATU-Eindhoven/Implementation) page.

1. All normalized GFP emissions are signals measured at 512 nm and normalized on the mCherry emission at 610 nm.

##### **References**

1. Daeffler, K., Galley, J., Sheth, R., Ortiz‐Velez, L., Bibb, C., Shroyer, N., Britton, R. and Tabor, J., 2017. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, [online] 13(4), p.923.
2. Bourdeau R, Lee-Gosselin A, Lakshmanan A, Farhadi A, Kumar S, Nety S et al. Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian hosts. Nature. 2018;553(7686):86-90.

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###### IBDetection

Non-Invasive Monitoring of IBD using Genetically Engineered Bacteria as Living Sensors.

###### Project

[Description](https://2021.igem.org/Team%3ATU-Eindhoven/Description)

[Lab](https://2021.igem.org/Team%3ATU-Eindhoven/Results)

[Model](https://2021.igem.org/Team%3ATU-Eindhoven/Model)

[Human Practices](https://2021.igem.org/Team%3ATU-Eindhoven/Human_Practices)

###### About Us

[Team](https://2021.igem.org/Team%3ATU-Eindhoven/Team)

[Partnership](https://2021.igem.org/Team%3ATU-Eindhoven/Partnership)

[Attributions](https://2021.igem.org/Team%3ATU-Eindhoven/Attributions)

[Sponsors](https://2021.igem.org/Team%3ATU-Eindhoven/Sponsors)

###### Contact

PO-box 513, 5600MB Eindhoven, Netherlands

igem@tue.nl

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[iGEM TU Eindhoven](https://2021.igem.org/Team%3ATU-Eindhoven)