iGEM 2021 — Team Bonn-Rheinbach

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

# Team:Bonn-Rheinbach/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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

# Team:Bonn-Rheinbach/Engineering

iGEM Bonn 2021 Biolan

[iGEM Bonn 2021](https://2021.igem.org/Team%3ABonn-Rheinbach)

[Project](https://2021.igem.org/Team%3ABonn-Rheinbach/Project)
[Social Outreach](https://2021.igem.org/Team%3ABonn-Rheinbach/Social_outreach)
[Attributions](https://2021.igem.org/Team%3ABonn-Rheinbach/Attributions)
[Team](https://2021.igem.org/Team%3ABonn-Rheinbach/Team)

![Architecture](https://static.igem.org/mediawiki/2021/9/93/T--Bonn-Rheinbach--Project_header.png)

# **Project**

# Engineering Cycle

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### Advancing through trial and error

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To tailor the process to industrial demands (e.g. potential for upscaling, low process costs, mild conditions), the approach shown in Figure 1 was first designed. It should initiate with the combination of REE-containing ore leachate and recombinant LanM purified from E. coli cell culture. At pH > 4.0, a LanM-REE complex form. By salting out with the kosmotropic salt ammonium sulfate, the complex precipitates from the leachate. Salting out is used to avoid costly affinity chromatography-based approaches which additionally might be sensitive to the highly complex ore leachate matrix. After removal of the REE-free supernatant and dissolving of the pellet (LanM-REE complex), the REE cations are released from the complex by acidification of the solution (pH < 1.5). Applying the acidic solution to an ultrafiltration unit with a molecular weight cut-off value of 3 kDa, apo-LanM (retentate/filter cake) is separated from highly pure REEs (flow-through). Apo-LanM is to be reused for further extraction cycles.

![Version1](https://static.igem.org/mediawiki/2021/5/53/T--Bonn-Rheinbach--E1Diagram.png)

---

![somepic](https://static.igem.org/mediawiki/2021/2/2a/T--Bonn-Rheinbach--E2Diagram.png)

Salting out with ammonium sulfate requires a high concentration of salt to obtain precipitation of the LanM-REE complex. In a LanM-added extraction run, a pellet formed after 1h incubation with 100% ammonium sulfate (750 mg to 1 ml solution). To verify that this precipitation was indeed based on the salting-out of the LanM-REE complex, another extraction run was performed without LanM (no-protein control). After adding ammonium sulfate to 100% solubility, a precipitate with a similar texture and pellet size was observed again. Thus, we assume that the high concentration of ammonium sulfate does not lead to a salting-out of the LanM-REE complex but instead results in the undesired precipitation of mineral salts. Thus, the process selectivity was not ensured and another option for isolation of the LanM-REE complex was chosen.

After redesigning the REE extraction process (Figure 2), the salting-out-based isolation of the LanM-REE complex was replaced by an ultrafiltration step. This should separate the solution into ore leachate (flow-through) and the LanM-REE complex (retentate). Since the other process parts showed no unsuitability, no further modifications were made.

---

After running the redesigned process, 63% of the initially applied REEs were obtained in the flow-through of ultrafiltration 2 (UF2-FT) – showing first promising results. To investigate if this modified approach relied on the REE-chelating activity of LanM, a no-protein control run was performed again. The process was identical to the approach described in Figure 2, albeit without the addition of recombinant LanM. As shown in Figure 11, the protein-free control run (w/o LanM) yielded only a neglectable amount of REEs in the UF2-FT fraction (2.7 nmol) while the LanM-containing process run (w/ LanM) resulted in the presence of 502 nmol of REEs in the desired fraction (UF2-FT). Thus, it can be solidly stated that LanM contributed to the enrichment of REEs in the final solution.

Overall, an initial engineering design cycle revealed the necessity to improve the designed process with respect to extraction selectivity. After identifying the critical step that reduced process specificity, the approach was modified. The redesigned process was run, including quality controls, and showed improved performance considering the selectivity of REE extraction.

![somepic](https://static.igem.org/mediawiki/2021/7/73/T--Bonn-Rheinbach--E3Diagram.png)

*written by Friedrich Ehinger*

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

# Team:Bonn-Rheinbach/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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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

# Team:Bonn-Rheinbach/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)