iGEM 2019 — Team Hamburg

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# Team:Hamburg/Notebook

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* [Home](https://2019.igem.org/Team%3AHamburg)
* Team

  + [Team Members](https://2019.igem.org/Team%3AHamburg/Team)
  + [Collaborations](https://2019.igem.org/Team%3AHamburg/Collaborations)
  + [Sponsors](https://2019.igem.org/Team%3AHamburg/Sponsors)
  + [Attributions](https://2019.igem.org/Team%3AHamburg/Attributions)
* Project

  + [Description](https://2019.igem.org/Team%3AHamburg/Description)
  + [Design](https://2019.igem.org/Team%3AHamburg/Design)
  + [Protocols](https://2019.igem.org/Team%3AHamburg/Protocols)
  + [Notebook](https://2019.igem.org/Team%3AHamburg/Notebook)
  + [Model](https://2019.igem.org/Team%3AHamburg/Model)
  + [Results](https://2019.igem.org/Team%3AHamburg/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AHamburg/Demonstrate)
* Parts

  + [Overview](https://2019.igem.org/Team%3AHamburg/Overview)
  + [Description](https://2019.igem.org/Team%3AHamburg/Part_Description)
  + [Basic](https://2019.igem.org/Team%3AHamburg/Basic_Part)
  + [Composite](https://2019.igem.org/Team%3AHamburg/Composite_Part)
  + [Part Collection](https://2019.igem.org/Team%3AHamburg/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AHamburg/Safety)
* Human Practices

  + [Integrated Human Practices](https://2019.igem.org/Team%3AHamburg/Human_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHamburg/Public_Engagement)
  + [World Map](https://2019.igem.org/Team%3AHamburg/world_map)
* Awards

  + [Integrated Human Practices](https://2019.igem.org/Team%3AHamburg/Human_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHamburg/Public_Engagement)
  + [Model](https://2019.igem.org/Team%3AHamburg/Model)
* [Judging Form ⇗](https://igem.org/2019_Judging_Form?team=Hamburg)

![First slide](https://static.igem.org/mediawiki/2019/6/64/T--Hamburg--ExploreFlagResearch2.jpg)

![Second slide](https://static.igem.org/mediawiki/2019/a/a4/T--Hamburg--BrilleBuchResearch2.jpg)

![Third slide](https://static.igem.org/mediawiki/2019/2/2d/T--Hamburg--BuchPenHandResearch2.jpg)

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# Notebook

 [GGA-Legend](https://static.igem.org/mediawiki/2019/6/6c/T--Hamburg--Notebook--GGALegende.pdf)

1. July
2. August
3. September
4. Ocotber

### July

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
| [1](https://static.igem.org/mediawiki/2019/f/f5/T--Hamburg--Notebook--190701.pdf) | [2](https://static.igem.org/mediawiki/2019/e/e4/T--Hamburg--Notebook--190702.pdf) | [3](https://static.igem.org/mediawiki/2019/d/da/T--Hamburg--Notebook--190703.pdf) | [4](https://static.igem.org/mediawiki/2019/2/2d/T--Hamburg--Notebook--190704.pdf) | [5](https://static.igem.org/mediawiki/2019/2/23/T--Hamburg--Notebook--190705.pdf) | [6](https://static.igem.org/mediawiki/2019/0/09/T--Hamburg--Notebook--190706.pdf) | [7](https://static.igem.org/mediawiki/2019/c/c0/T--Hamburg--Notebook--190707.pdf) |
| [8](https://static.igem.org/mediawiki/2019/5/5a/T--Hamburg--Notebook--190708.pdf) | [9](https://static.igem.org/mediawiki/2019/e/e3/T--Hamburg--Notebook--190709.pdf) | [10](https://static.igem.org/mediawiki/2019/6/60/T--Hamburg--Notebook--190710.pdf) | [11](https://static.igem.org/mediawiki/2019/0/00/T--Hamburg--Notebook--190711.pdf) | 12 | [13](https://static.igem.org/mediawiki/2019/2/29/T--Hamburg--Notebook--190713.pdf) | 14 |
| [15](https://static.igem.org/mediawiki/2019/e/e6/T--Hamburg--Notebook--190715.pdf) | 16 | [17](https://static.igem.org/mediawiki/2019/a/af/T--Hamburg--Notebook--190717.pdf) | [18](https://static.igem.org/mediawiki/2019/a/aa/T--Hamburg--Notebook--190718.pdf) | 19 | 20 | 21 |
| [22](https://static.igem.org/mediawiki/2019/f/f9/T--Hamburg--Notebook--190722.pdf) | 23 | [24](https://static.igem.org/mediawiki/2019/e/e3/T--Hamburg--Notebook--190724.pdf) | [25](https://static.igem.org/mediawiki/2019/0/0b/T--Hamburg--Notebook--190725.pdf) | [26](https://static.igem.org/mediawiki/2019/d/d7/T--Hamburg--Notebook--190726.pdf) | 27 | 28 |
| [29](https://static.igem.org/mediawiki/2019/b/b8/T--Hamburg--Notebook--190729.pdf) | 30 | 31 |  |  |  |  |

### August

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |

  |

  |

 [1](https://static.igem.org/mediawiki/2019/6/6a/T--Hamburg--Notebook--190801.pdf) |

 2 |

 3 |

 [4](https://static.igem.org/mediawiki/2019/5/5d/T--Hamburg--Notebook--190804.pdf) |

| [5](https://static.igem.org/mediawiki/2019/5/50/T--Hamburg--Notebook--190805.pdf) |

 [6](https://static.igem.org/mediawiki/2019/7/73/T--Hamburg--Notebook--190806.pdf) | [7](https://static.igem.org/mediawiki/2019/f/f3/T--Hamburg--Notebook--190807.pdf) | 8 | [9](https://static.igem.org/mediawiki/2019/0/05/T--Hamburg--Notebook--190809.pdf) | 10 | 11 || 12 | 13 | 14 | [15](https://static.igem.org/mediawiki/2019/6/62/T--Hamburg--Notebook--190815.pdf) | 16 | 17 | 18 |
| [19](https://static.igem.org/mediawiki/2019/5/5e/T--Hamburg--Notebook--190817.pdf) | 20 | [21](https://static.igem.org/mediawiki/2019/6/61/T--Hamburg--Notebook--190821.pdf) | [22](https://static.igem.org/mediawiki/2019/5/57/T--Hamburg--Notebook--190822.pdf) | [23](https://static.igem.org/mediawiki/2019/7/74/T--Hamburg--Notebook--190823.pdf) | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 | 31 |  |

### September

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | [2](https://static.igem.org/mediawiki/2019/d/dd/T--Hamburg--Notebook--190902.pdf) | [3](https://static.igem.org/mediawiki/2019/d/d1/T--Hamburg--Notebook--190903.pdf) | [4](https://static.igem.org/mediawiki/2019/4/4a/T--Hamburg--Notebook--190904.pdf) | [5](https://static.igem.org/mediawiki/2019/9/91/T--Hamburg--Notebook--190905.pdf) | [6](https://static.igem.org/mediawiki/2019/d/d4/T--Hamburg--Notebook--190906.pdf) | [7](https://static.igem.org/mediawiki/2019/c/c8/T--Hamburg--Notebook--190907.pdf) |
| 8 | [9](https://static.igem.org/mediawiki/2019/e/ee/T--Hamburg--Notebook--190909.pdf) | [10](https://static.igem.org/mediawiki/2019/a/a3/T--Hamburg--Notebook--190910.pdf) | [11](https://static.igem.org/mediawiki/2019/0/0c/T--Hamburg--Notebook--190911.pdf) | 12 | [13](https://static.igem.org/mediawiki/2019/d/d6/T--Hamburg--Notebook--190913.pdf) | [14](https://static.igem.org/mediawiki/2019/9/9a/T--Hamburg--Notebook--190914.pdf) |
| [15](https://static.igem.org/mediawiki/2019/0/0d/T--Hamburg--Notebook--190915.pdf) | [16](https://static.igem.org/mediawiki/2019/a/ae/T--Hamburg--Notebook--190916.pdf) | [17](https://static.igem.org/mediawiki/2019/7/7b/T--Hamburg--Notebook--190917.pdf) | [18](https://static.igem.org/mediawiki/2019/c/c8/T--Hamburg--Notebook--190918.pdf) | [19](https://static.igem.org/mediawiki/2019/3/38/T--Hamburg--Notebook--190919.pdf) | [20](https://static.igem.org/mediawiki/2019/f/fa/T--Hamburg--Notebook--190920.pdf) | 21 |
| [22](https://static.igem.org/mediawiki/2019/0/03/T--Hamburg--Notebook--190922.pdf) | [23](https://static.igem.org/mediawiki/2019/9/90/T--Hamburg--Notebook--190923.pdf) | [24](https://static.igem.org/mediawiki/2019/e/e8/T--Hamburg--Notebook--190924.pdf) | [25](https://static.igem.org/mediawiki/2019/1/1f/T--Hamburg--Notebook--190925.pdf) | [26](https://static.igem.org/mediawiki/2019/f/f8/T--Hamburg--Notebook--190926.pdf) | [27](https://static.igem.org/mediawiki/2019/a/aa/T--Hamburg--Notebook--190927.pdf) | [28](https://static.igem.org/mediawiki/2019/d/dc/T--Hamburg--Notebook--190928.pdf) |
| 29 | 30 | 31 |  |  |  |  |

### October

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| [16](https://2019.igem.org/File%3AT--Hamburg--191016.pdf) | 17 | 18 | 19 | [20](https://2019.igem.org/File%3AT--Hamburg--191020.pdf) | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 |  |  |  |  |  |  |

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![Universität Hamburg](https://static.igem.org/mediawiki/2019/f/f2/T--Hamburg--sponsor_UniHamburg.jpg)
![Jung Stiftung](https://static.igem.org/mediawiki/2019/d/d5/T--Hamburg--sponsor_jungstiftung.jpg)
![Altona Diagonstics](https://static.igem.org/mediawiki/2019/8/86/T--Hamburg--Sponsor_Altona.jpg)
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![Biomol](https://static.igem.org/mediawiki/2019/6/64/T--Hamburg--Sponsor_Biomol.jpg)
![Claussen Simons](https://static.igem.org/mediawiki/2019/d/d9/T--Hamburg--sponsor_clausenstiftung.jpg)
![MLP](https://static.igem.org/mediawiki/2019/6/63/T--Hamburg--Sponsor_MLP.jpg)
![Pohl Boskamp](https://static.igem.org/mediawiki/2019/e/e8/T--Hamburg--sponsor_PohlBoskamp.jpg)
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[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Hamburg/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Hamburg/Results

[![Logo](https://static.igem.org/mediawiki/2019/8/81/T--Hamburg--ueberschrift_ribbot.png)](https://2019.igem.org/Team%3AHamburg)

* [Home](https://2019.igem.org/Team%3AHamburg)
* Team

  + [Team Members](https://2019.igem.org/Team%3AHamburg/Team)
  + [Collaborations](https://2019.igem.org/Team%3AHamburg/Collaborations)
  + [Sponsors](https://2019.igem.org/Team%3AHamburg/Sponsors)
  + [Attributions](https://2019.igem.org/Team%3AHamburg/Attributions)
* Project

  + [Description](https://2019.igem.org/Team%3AHamburg/Description)
  + [Design](https://2019.igem.org/Team%3AHamburg/Design)
  + [Protocols](https://2019.igem.org/Team%3AHamburg/Protocols)
  + [Notebook](https://2019.igem.org/Team%3AHamburg/Notebook)
  + [Model](https://2019.igem.org/Team%3AHamburg/Model)
  + [Results](https://2019.igem.org/Team%3AHamburg/Results)
  + [Demonstrate](https://2019.igem.org/Team%3AHamburg/Demonstrate)
* Parts

  + [Overview](https://2019.igem.org/Team%3AHamburg/Overview)
  + [Description](https://2019.igem.org/Team%3AHamburg/Part_Description)
  + [Basic](https://2019.igem.org/Team%3AHamburg/Basic_Part)
  + [Composite](https://2019.igem.org/Team%3AHamburg/Composite_Part)
  + [Part Collection](https://2019.igem.org/Team%3AHamburg/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AHamburg/Safety)
* Human Practices

  + [Integrated Human Practices](https://2019.igem.org/Team%3AHamburg/Human_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHamburg/Public_Engagement)
  + [World Map](https://2019.igem.org/Team%3AHamburg/world_map)
* Awards

  + [Integrated Human Practices](https://2019.igem.org/Team%3AHamburg/Human_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHamburg/Public_Engagement)
  + [Model](https://2019.igem.org/Team%3AHamburg/Model)
* [Judging Form ⇗](https://igem.org/2019_Judging_Form?team=Hamburg)

![First slide](https://static.igem.org/mediawiki/2019/6/64/T--Hamburg--ExploreFlagResearch2.jpg)

![Second slide](https://static.igem.org/mediawiki/2019/a/a4/T--Hamburg--BrilleBuchResearch2.jpg)

![Third slide](https://static.igem.org/mediawiki/2019/2/2d/T--Hamburg--BuchPenHandResearch2.jpg)

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# Results

The aim of this project was to enable a transformation of three plasmids into one bacterium with only one antibiotic resistance instead of three. The idea is based on the paper “Complex cellular logic computation using ribocomputing devices” by Green et al. ([Nature, 2017](%E2%80%9Dhttps%3A//www.nature.com/articles/nature23271%E2%80%9D)). In our project we focused on an AND logic, which means that two or more so called trigger need to be present at a time to enable gene expression. The strategy is based on toehold switches that are forming hairpin structures, which hide the ribosome binding site and the start codon. Those so called gates can be opened by the triggers due to a higher binding affinity.
To read more about the details visit our [Description and [Design pages.

## Promoter characterization

We characterized the activity of the promoters we used in our project, the two constitutive promoters from the Anderson Family, BBa\_J23100 and BBa\_J23102. We measured the expression of GFP downstream of the promoters.
The parts were cloned into *E. coli* DH5α competent cells. The fluorescence measurements were performed using flow cytometry and plate reader measurements. The results of the flow cytometry measurement are shown in figure 1.

![](https://static.igem.org/mediawiki/2019/5/5a/T--Hamburg--ResultsFigure1.jpeg)

**Figure 1:** FACS measurement of GFP in *E. coli* with the promoters BBa\_J23100 and BBa\_J23102. Non-competent cells without GFP as negative control show a background fluorescence signal (orange); competent cells with GFP and the different promoters BBa\_J23100 (green and pink) and BBA\_J23102 (blue) show clear fluorescence signals with different intensities depending on the promoter.

The results confirm the different strengths of the constitutive promoters. BBa\_J23100 expresses a stronger fluorescence signal than BBa\_J23102. As a negative control we used non-competent cells to measure background emission.
The data used to calibrate the plate reader measurements are shown in figure 2. The Excel file used for the calibration can be viewed [here.](https://static.igem.org/mediawiki/2019/1/18/T--Hamburg--ResultsFigure10.xlsx)
The results of our measurement of GFP on the plate reader are shown in figure 3.
The corresponding Excel file can be found [here.](https://static.igem.org/mediawiki/2019/a/a2/T--Hamburg--ResultsFigure8.xlsx%29)

![](https://static.igem.org/mediawiki/2019/8/8a/T--Hamburg--ResultsFigure9.png)

**Figure 2:** Plate reader calibration. A) Particle standard curve. B) Particle standard curve in log scale. C) Fluorescein standard curve. D) Fluorescein standard curve in log scale.

![](https://static.igem.org/mediawiki/2019/d/d4/T--Hamburg--ResultsFigure7.png
)

**Figure 3:** Plate reader measurement of GFP in *E. coli* with the promoters BBa\_J23100 and BBa\_J23102. Promoter BBa\_J23100 (green) expresses higher fluorescence than BBa\_J23102 (blue). As control non-competent cells were used (orange). A) Fluorescence calculated in MFEL/particle B) Fluorescence calculated in µM Flourescein/OD600. Error bars show the standard deviation.

The results confirm the previous results from the FACS measurement. The promoter BBa\_J23100 expresses a stronger fluorescence signal when compared to BBa\_J23102.
We calculated the strengths of these promoters relative to the control:

* BBa\_J23100: 2.28
* BBa\_J23102: 1.84

The relative strength of BBa\_J23102 when compared to BBa\_J23100 is 0.8. In comparison, the values in the registry is 0.856.
Additionally, we sequenced the plasmids used in our measurement. The reported sequence matched the Anderson promoter BBa\_J23100. The reported sequence for the promoter BBa\_J23102 showed three point mutations, which could have impacted the strength of the promoter. The following mutations occurred: 6A>G, 22G>A, 27T>A. This explains the decrease in the relative promoter strength of BBa\_J23100 compared to BBa\_J23102.

## Gate and Trigger

We tested the gate plasmid with our two trigger plasmids by performing simultaneous transformations with these plasmids. After transformation we selected the bacteria by plating on LB-agar plates with chloramphenicol. We compared the results with positive and negative controls, using the empty pSB1C3 backbone and pSB1A3 respectively.
The result of the transformation with all three plasmids after selection with chloramphenicol is shown in figure 4.

![]( https://static.igem.org/mediawiki/2019/4/48/T--Hamburg--ResultsFigure5.jpeg)

**Figure 4:**  *E. coli* cells transformed with three plasmids, the plasmid carrying the gate, the plasmid carrying trigger one and the plasmid carrying trigger two. The cells were plated on LB-agar plates with chloramphenicol, bacterial growth was observed.

Colonies grew on chloramphenicol plates after transformation with all three plasmids. This shows that our RNA based logic circuit allows the expression of the antibiotic resistance. No colonies grew on the plates with the negative control. Colonies also grew on the plates with the positive control.
To further test the trigger and the gate, we performed transformations with each plasmid individually. The results are shown in figure 5.

![](https://static.igem.org/mediawiki/2019/e/ec/T--Hamburg--ResultsFigure4.jpeg)

**Figure 5:**  Cell plated out on chloramphenicol agar plates. Left: cells transformed with the gate plasmid. Middle: cells transformed with the trigger 1 plasmid. Right: cells transformed with the trigger 2 plasmid.

Unfortunately colonies also grew on the plates with just the individual transformation of the gate plasmid, though the number of colonies was much lower when compared to the transformation with all three plasmids. We sequenced the gate plasmid to confirm correct assembly. The results showed no mutation in the gate sequence. We concluded that the gate is showing signs of leakage, expressing the antibiotic resistance even in the absence of triggers. This basal expression was rather strong due to the strong promoter we used for our experiments.
To measure the gates leakage, we observed cells transformed with all three plasmids on plates with different chloramphenicol concentrations and compared them to cells transformed with only the gate plasmid. As a positive control we used the pSB1C3 backbone. The Excel file used for the calibration can be viewed [here.](https://static.igem.org/mediawiki/2019/c/c3/T--Hamburg--ResultsFigure6.xlsx) The number of colonies per plate after twelve hours of incubation is shown in figure 6.

![](https://static.igem.org/mediawiki/2019/b/bd/T--Hamburg--ResultsFigure3.jpeg)

**Figure 6:**  Number of colonies per plate depending on the chloramphenicol concentration. *E. coli* DH5α cells were transformed with the gate plasmid (blue), the gate plasmid together with both trigger plasmids (red), or with the control backbone pSB1C3 (green). The colonies were counted after 12 hours.

The data clearly shows that the gate is leaking, but when compared to the triple transformation (red) and the control (green) the number of colonies when transformed with only the gate plasmid was significantly lower. Additionally the number of colonies when transformed with all three plasmids was larger than when transformed with the positive control.

We also used a plate reader to measure the growth rate of cells that were transformed with three different plasmids (pSB1A3, pSB1C3, pSB1K3 or BBa\_K2970003, BBa\_K2970004, BBa\_K2970006) and compared it to cells without any plasmids. The results are shown in figure 7.

![](https://static.igem.org/mediawiki/2019/7/7e/T--Hamburg--ResultsFigure11.png)

**Figure 7:**  Plate reader growth curves of *E. coli* DH5α. The generation time was measured and calculated for non competent cells (orange), for cells transformed with the backbones pSB1A3, pSB1C3 and pSC1K3.M1 (blue), and for cells transformed with our two trigger plasmids together with the gate plasmid (green). Error bars show the standard deviation.

For cells transformed with three plasmids an impaired growth rate can be observed.
We could not show that the transformation of our three plasmids was less harmful to the bacteria than the transformation with three different antibiotics, instead it shows comparable levels of stress.

Due to the linker between the gate and our gene of interest (chloramphenicol acetyl transferase) additional bases were attached to the gene which might affect the functionality of the protein as it contained an additional 19 amino acids. To test the genes activity we inserted the sequence containing the additional bases into pSB1A3, transformed it into bacteria and plated the cells on chloramphenicol plates. The results of this experiment are shown in figure 8.

![](https://static.igem.org/mediawiki/2019/2/2a/T--Hamburg--ResultsFigure2.jpeg)

**Figure 8:**  Test transformation to check chloramphenicol resistance gene. *E. coli* DH5α cells were transformed with a test plasmid containing the modified chloramphenicol resistance gene (A). For negative control (B) pSB1A3 was used. For positive control (C) pSB1C3 was used.

Several colonies grew on the plate with bacteria transformed with the modified chloramphenicol acetyl transferase. The negative control showed no cell growth while on the plate with the positive control a cell turf has grown. This confirms that the activity of the chloramphenicol acetyl transferase was not significantly impaired by the additional amino acids.](%E2%80%9Dhttps%3A//2019.igem.org/Team%3AHamburg/Design%E2%80%9D)](%E2%80%9Dhttps%3A//2019.igem.org/Team%3AHamburg/Discription%E2%80%9D)

![Universität Hamburg](https://static.igem.org/mediawiki/2019/f/f2/T--Hamburg--sponsor_UniHamburg.jpg)
![Jung Stiftung](https://static.igem.org/mediawiki/2019/d/d5/T--Hamburg--sponsor_jungstiftung.jpg)
![Altona Diagonstics](https://static.igem.org/mediawiki/2019/8/86/T--Hamburg--Sponsor_Altona.jpg)
![BioLabs](https://static.igem.org/mediawiki/2018/1/13/T--Hamburg--Sponsors_NEB.svg)
![Biomol](https://static.igem.org/mediawiki/2019/6/64/T--Hamburg--Sponsor_Biomol.jpg)
![Claussen Simons](https://static.igem.org/mediawiki/2019/d/d9/T--Hamburg--sponsor_clausenstiftung.jpg)
![MLP](https://static.igem.org/mediawiki/2019/6/63/T--Hamburg--Sponsor_MLP.jpg)
![Pohl Boskamp](https://static.igem.org/mediawiki/2019/e/e8/T--Hamburg--sponsor_PohlBoskamp.jpg)
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