iGEM 2021 — Team NAWI-Graz

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

# Team:NAWI-Graz/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:NAWI-Graz/Engineering

Team:NAWI-Graz - 2021.igem.org

# Team:NAWI-Graz

[Home](https://2021.igem.org/Team%3ANAWI-Graz)

Project

[Description](https://2021.igem.org/Team%3ANAWI-Graz/Description)
[Contribution](https://2021.igem.org/Team%3ANAWI-Graz/Contribution)
[Model](https://2021.igem.org/Team%3ANAWI-Graz/Model)
[Excellence in another Area](https://2021.igem.org/Team%3ANAWI-Graz/excellence)

Team

[Members](https://2021.igem.org/Team%3ANAWI-Graz/Members)
[Collaborations](https://2021.igem.org/Team%3ANAWI-Graz/Collaborations)

Research

[Engineering](https://2021.igem.org/Team%3ANAWI-Graz/Engineering)
[Implementation](https://2021.igem.org/Team%3ANAWI-Graz/Implementation)

Human Practice

[Human Practices](https://2021.igem.org/Team%3ANAWI-Graz/Human_Practices)
[Sponsors](https://2021.igem.org/Team%3ANAWI-Graz/Sponsors)
[Attributions](https://2021.igem.org/Team%3ANAWI-Graz/Attributions)

Media

[Promotion Video](https://2021.igem.org/Team%3ANAWI-Graz/Promotion-Video)
[Presentation Video](https://2021.igem.org/Team%3ANAWI-Graz/Presentation-Video)

# Engineering

After we decided to tackle the phosphate problem, we thought of ways to build a system that would be easy to deploy and that utilises bacteria that are already present in agricultural soil.
Our approach included making phosphate more available to plants. For this purpose we came up with an approach to use the quorum sensing system of the rhizosphere inhabiting Bacteria.

## BBa\_K3928000

### Design:

For the realization of our vision we needed to adapt our plasmid construct to an already existing system in E. coli. The phoR - phosphate regulon sensor protein is a membrane-associated protein that phosphorylates and thus activates phoB-proteins in response to limited environmental phosphate concentrations. As part of phoB-activation, a multitude of regulative operons associated to the phosphate metabolic pathway also get activated, among them the alkaline phosphatase promoter PphoA.

The reason we used this promoter specifically is that previous IGEM-competitors characterized its usage adequately for us to modify it to the degree we hoped for. In the course of our initial research period our original planned implementation for the negative regulation of PphoA in response to environmental phosphate concentrations posed a substantial problem: since the promoter activates at low and deactivates at high phosphate levels our system would not be able to fulfill its intended use of operating as soon as fertilizer (which contains phosphorus) is added. So we had to design a system.
![](https://static.igem.org/mediawiki/2021/0/08/T--NAWI-Graz--engineering_foto_01.jpg)

## Build:

Our goal was it to built a plasmid for E.coli detection by a system in which PhoB, which is native to E. coli binds to the used PphoA promoter (BBa\_K1139200) activating transcription of iLacI (BBa\_K3257045) which can bind to the LacI/LacO binding site (BBA\_K3257066). By that, it prevents the expression of eGFP (BBa\_K1911005) which is regulated via the constitutive promoter (BBa\_J23106).

![](https://static.igem.org/mediawiki/2021/5/5f/T--NAWI-Graz--engineering_01.png)

At low phosphate concentrations, the PphoA promoter is active, the LacI protein is expressed, which leads to a repression of the constitutive promoter via the LacO operating site. Thus no GFP is expressed.

![](https://static.igem.org/mediawiki/2021/3/33/T--NAWI-Graz--engineering_02.png)

At high phosphate concentrations, the PphoA promoter is inactive, the LacI protein does not get expressed, therefore GFP can be expressed due to the usage of a constitutive promoter.

We used the iGEM plasmid BBa\_J04450 as the vector for our first attempt. E. coli Top 10 cells were transformed with our plasmid utilising the RFP expression of this plasmid as a marker for a successful transformation. After the transformation was completed, colonies were visible. The vector was extracted via miniprep.

The two insert parts, one with the PhoA promoter (BBa\_K1139200), the LacI (BBa\_K3257045) gene and the LacL/LacO binding site (BBA\_K3257066), and the second one with the eGFP (BBa\_K1911005) marker and a constitutive promoter (BBa\_J23106) were synthesised. To have enough DNA material the two parts got separately amplified by PCR.

The construction of the BBa\_K3928000 plasmid was done by restriction digestion and ligation. The PCR product of part 1 was digested with EcoRI and BamHI and the product of part 2 was digested with BamHI and PstI. The vector was digested with EcoRI and PstI. After this we checked the digestion of the parts by gel electrophoresis.

The bands of interest containing the digested parts were cut out of the gel and extracted. After checking for the desired parts, we ligated the 2 fragments described above as part 1 and part 2 with the vector.

The result was plasmid BBa\_K3928000

![](https://static.igem.org/mediawiki/2021/f/f1/T--NAWI-Graz--description_foto_03.jpg)

## Test

To assess whether our construct was functional we decided to do an assay as previous iGEM teams suggested [[1](#references)]. As previously mentioned under the presence of phosphate our construct BBa\_K3928000 should express GFP. Further we used various different concentrations of K2HPO4 as our phosphate source, to determine a signal threshold for excitation.

We started with an ONC of our E. coli TOP10 cells with BBa\_K3928000 in MOPS minimal media [[2](#references)] with phosphate (0.132 M K2HPO4) and chloramphenicol (25µg/ml) at 37°C. After diluting the cells to an OD600 of 0.1 in fresh MOPS media we incubated them further. Then the cells were centrifuged and washed twice with fresh MOPS without phosphate and resuspended to an OD600 of 10.

To determine the concentration necessary for eGFP excitation, 300µl of cells were incubated at 26°C with different concentrations (4µM, 5µM, 10µM, 50µM, 100µM, 1000µM) of K2HPO4 in MOPS media with chloramphenicol (25µg/ml). After 120 minutes, fluorescence intensity was measured.

![](https://static.igem.org/mediawiki/2021/6/67/T--NAWI-Graz--engineering_foto_02.jpg)

## Learn:

The Phosphate Assay did not provide a valid result. Therefore we were not able to confirm the functioning of our part (BBa\_K3928000).

The photon emission of the expressed GFP should have been measured in a fluorimeter. After stimulation with light at the excitation wavelength, no emission at the target wavelength could be measured. We could not assess what the reason for that was. It is possible that the plasmid for the expression of the GFP was not inserted into the cells properly with the transformation. The phosphate concentrations in the assay could have been to low, or the plasmid was not abundant in the cells in high enough numbers to produce enough GFP for a measurement.

## BBa\_3928003

### Design:

This plasmid was created with the basis of the BBa\_K3928000 plasmid, by exchanging the eGFP of the positive-phosphate-phoA-GFP-sensor (BBa\_3928002) by the autoinducer synthetase for AHL (BBa\_C0061)
This, then, should lead to the excretion of AHLs as quorum sensing molecules. The cell containing BBa\_3928003 thereby works as a sender cell that reacts to phosphate akkumulation. The receiver cell should react to these AHLs by expressing marker genes like GFP.

![](https://static.igem.org/mediawiki/2021/2/2e/T--NAWI-Graz--engineering_03.png)

At low phosphate concentrations, the PphoA promoter is active, the LacI protein is expressed, which leads to a repression of the constitutive promoter via LacO and no expression of the AHL synthase. Thus no quorum sensing molecule 3OC6HSL gets produced. In this scenario, the receiver cell perceives no quorum sensing molecules and should not react in any way.

![](https://static.igem.org/mediawiki/2021/8/8e/T--NAWI-Graz--engineering_04.png)

At high phosphate concentrations, the PphoA promoter is inactive, the LacI protein does not get expressed.This leads, as in BBa\_K3928000 described, to the expression of the AHL synthetase which produces quorum sensing molecules (3OC6HSL). This should then be detected by the receiver cells, which should express a marker molecule in response to the quorum sensing molecules.

![](https://static.igem.org/mediawiki/2021/3/38/T--NAWI-Graz--description_foto_02.jpg)

## Build:

To generate BBa\_3928003, we tried to use the Gibson cloning method.

Two sets of primers were designed for the gibson assembly. The first one amplified the LuxI gene of interest with the additional assembly overhangs.

The second one amplified the vector constructed from BBa\_K3928000 but without eGFP.
If properly assembled via gibson assembly, the created plasmid then should be brought into E.coli via transformation. The resulting transformed cell should then excrete the quorum sensing molecules described above.

Unfortunately the gibson assembly was not successful which denied us from going on further in our research.

## References

[1] Marie-Andre´e Dollard, Patrick Billard; Whole-cell bacterial sensors for the monitoring of phosphate bioavailability, 2013
[2]Frederick C. Neidhardt, Philip L. Bloch, David F. Smith;
Culture Medium for Enterobacteria, 1974

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

# Team:NAWI-Graz/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:NAWI-Graz/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)