iGEM 2024 — Team hannover

{% extends "layout\_notebook.html" %}
{% block title %}Lab Notebook: Documenting Our Scientific Journey{% endblock %}
{% block lead %}Welcome to the Lab Notebook of the Hydro Guardian. Dive into the detailed records of our experiments, methodologies, and daily progress as we navigate the exciting challenges of synthetic biology.{% endblock %}
{% block page\_content %}

# iGEM Team Hannover 2024: Lab-Notebooks

---

## Biological Experiments

In this section, we present a general overview of our performed biological experiments during the past months. For a detailed insight in our daily lab work feel free to take a look at the PDF version of our lab notebook below or at our [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

## Week of April 8th to 14th

* PCRs for genes of MTF1 ([BBa\_K5317007](https://parts.igem.org/Part%3ABBa_K5317007)) and MRE promoters (MREdada ([BBa\_K5317006](https://parts.igem.org/Part%3ABBa_K5317006)), MREa ([BBa\_K5317004](https://parts.igem.org/Part%3ABBa_K5317004)), MREwt ([BBa\_K5317003](https://parts.igem.org/Part%3ABBa_K5317003)), MREd ([BBa\_K5317005](https://parts.igem.org/Part%3ABBa_K5317005))).

## Week of April 15th to 21st

* PCRs for genes of ATF2 ([BBa\_K5317016](https://parts.igem.org/Part%3ABBa_K5317016)), CcpA ([BBa\_K5317014](https://parts.igem.org/Part%3ABBa_K5317014)) GraR ([BBa\_K5317015](https://parts.igem.org/Part%3ABBa_K5317015)) and MTF1.
* Prepared PCR reactions for metal-gene cloning (MTF-1, mRuby2 ([BBa\_K5317001](https://parts.igem.org/Part%3ABBa_K5317001))).
* Restriction digestion of pEGFP-C2 ([BBa\_K3338020](https://parts.igem.org/Part%3ABBa_K3338020)) plasmids for MRE promoter constructs using AseI and NheI enzymes.
* HiFi reactions of MRE promoter constructs (MREdada, MREa, MREnormal, MREd).
* Colony PCR and MiniPreps for analyzing MRE constructs.

## Week of April 22nd to 28th

* Sequencing of MRE promoter constructs.

## Week of April 29th to Mai 5th

* Repeated PCRs from gBlocks for the genes of PknB, CcpA, and GraR.

## Week of May 6th to 12th

* PCRs for genes of ATF2, CcpA, and GraR (mRuby2 constructs).
* Restriction digestion with DpnI of GraR and CcpA.
* Restriction digestion of pEGFP-C2 with BamHI and NheI for creating a backbone for HiFi reaction of CcpA, GraR and MTF1.
* HiFi reactions for various constructs:
  + PknB and pEGFP-C2 to composite part CMV-EGFP-PknB ([BBa\_K5317018](https://parts.igem.org/Part%3ABBa_K5317018)).
  + CcpA, mRuby2 and pEGFP-C2 to composite part CMV-CcpA-mRuby2 ([BBa\_K5317019](https://parts.igem.org/Part%3ABBa_K5317019)).
  + GraR, mRuby2 and pEGFP-C2 to composite part CMV-GraR-mRuby2 ([BBa\_K5317020](https://parts.igem.org/Part%3ABBa_K5317020)).
  + MTF1, mRuby2 and pEGFP-C2 to composite part CMV-MTF1-mRuby2 ([BBa\_K5317012](https://parts.igem.org/Part%3ABBa_K5317012)).
* Heat shock transformation of cloned HiFi products.
* Colony PCR for CcpA, GraR, and MTF-1 (mRuby2 constructs).

## Week of May 13th to 19th

* MiniPreps and sequencing of CcpA, GraR, and MTF1 (mRuby2 constructs).
* Restriction digestion of pEGFP-C2 with BamHI & SalI for PknB construct (repeated multiple times).
* Restriction digestion of pEGFP-C2 with EcoRI & NheI for MTF1 construct (repeated multiple times).

## Week of May 20th to 26th

* Repetition of HiFi reaction for CMV-EGFP-PknB and CMV-MTF1-mRuby2.
* Heat shock transformation of HiFi products.
* Colony PCRs, MiniPreps and sequencing for the composite parts.

## Week of May 27th to June 2nd

* HiFi reaction of ATF2, mRuby2 and pEGFP-C2 to composite part CMV-ATF2-mRuby2  ([BBa\_K5317021](https://parts.igem.org/Part%3ABBa_K5317021)).
* Heat shock transformation of HiFi product.
* Colony PCRs, MiniPreps and sequencing of the composite parts.
* Repetition of PCR for PknB with Phusion- and Q5-Polymerase, followed by another HiFi reaction for CMV-EGFP-PknB.
* Heat shock transformation and colony PCR, MiniPrep and sequencing for CMV-EGFP-PknB.

## Week of June 3rd to 9th

* Repetition of HiFi reaction and following Colony PCR of ten clones for CMV-EGFP-PknB.
* Success for one clone (K6) validated via gel electrophoresis.
* Mini-Prep & Sequencing of clone K6.

## Week of June 10th to 16th

* Further sequencing of K6 to validate correctness.
* Showed mutation in the sequence of PknB.
* Order of new gBlock of PknB.

## Week of June 17th to 23rd

* Thaw 2x 1 ml HEK293T P5 cells.
* Passaging of HEK293T cells.

## Week of June 24th to 30th

* First test transfection of HEK293T cells with CMV-MTF1-mRuby2 and pEGFP-C2.

## Week of July 1st to 7th

* Repetition of HiFi, colony PCR, MiniPrep and Sequencing ofCMV-EGFP-PknB with new PknB gBlock (successful).
* Qualitative validation of single MTF-1, MREwt, MREa, MREd and MREdada as well as the co-transfection of MTF-1 with all promoter constructs.
* Qualitative validation of single PknB, GraR, ATF2 and CcpA.

## Week of July 8th to 14th

* Kill curve of HEK293T cells under treatment with CuCl2 (0 µM, 50 µM, 250 µM, 500 µM, 1 mM, 2 mM) and ZnCl2 (0 µM, 50 µM, 250 µM, 500 µM, 1 mM, 5 mM).
* Qualitative validation of single ATF2 and CcpA.

## Week of July 15th to 21st

* Qualitative validation of PknB co-transfected with ATF2 and GraR.

## Week of July 22nd to 28th

* Kill curve of HEK293T cells under treatment with CuSO4 (0 µM, 50 µM, 250 µM, 500 µM, 1 mM, 2 mM).
* Qualitative validation of MTF-1 co-transfected with MREwt and MREdada promoters via fluorescent microscopy before and after stimulation with CuSO4.

## Week of July 29th to August 4th

* Qualitative validation of MTF-1 co-transfected with MREa and MREd promoters via fluorescent microscopy before and after stimulation with CuSO4.

## Week of August 5th to August 11th

* Restriction digestion of pEGFP-C2 with AseI & BamHI for creating a backbone for the HiFi reaction of ATF2.

## Week of August 12th to August 18th

* Quantitative FACS validation of metal constructs after stimulation with CuSO4.
* Cloning of ATF2\_miRFP-Promoter using the Parts miRFP670 ([BBa\_K5317002](https://parts.igem.org/Part%3ABBa_K5317002)) and ATF2-3xCre3xAP1-Promoter ([BBa\_K5317017](https://parts.igem.org/Part%3ABBa_K5317017)) via HiFi DNA Assembly and heat shock transformation in *E. coli* DH5-alpha.

## Week of August 19th to August 25th

* Repetition of ATF2\_miRFP-Promoter ([BBa\_K5317022](https://parts.igem.org/Part%3ABBa_K5317022)) cloning after failure in the previous week.
* Sequencing of correct ATF2\_miRFP-Promoter clone K7.

## Week of August 26th to September 1st

* Qualitative validation of antibiotic constructs via fluorescent microscopy.
* Kill curve of HEK293T cells under treatment with Ampicillin (0 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, 100 µg/mL, 500 µg/mL)
* Testing of different construct combinations (PknB + Promoter; ATF2 + Promoter; single Promoter; PknB + ATF2 + Promoter).

## Week of September 2th to September 8th

* Qualitative and Quantitative validation of antibiotic constructs via fluorescent microscopy and FACS analysis, respectively.

## Week of September 9th to September 15th

* Further qualitative validations of metal and antibiotic constructs via fluorescent microscopy.

## Physics Experiments

Here you find the notebook about the [Spectroscopic Analysis](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27spectroscopy%27%29%20%7D%7D) we performed in addition to the biological experiments.

[Back to the Top](#nb_top)

{% endblock %}

{% extends "layout\_engineering.html" %}
{% block title %}Engineering Our Path to Innovation{% endblock %}
{% block lead %}Dive into the engineering journey of the Hydro Guardian and discover how we've applied the iGEM Engineering Cycle to develop groundbreaking solutions in synthetic biology.{% endblock %}
{% block page\_content %}

* ![](https://static.igem.wiki/teams/5317/logos/hg-gespiegelt.png)

  [**β-Lactam Detection Unit**: Developed through the iGEM engineering cycle to use the **PASTA domain** to detect **beta-lactam antibiotics** with a strong fluorescent signal.](#betaLactamDU)
* ![](https://static.igem.wiki/teams/5317/logos/hg-gespiegelt.png)

  [**Metal Detection Unit**: Refined through the iGEM engineering cycle to improve the performance of **Metal-Responsive Elements (MREs)** for accurate detection of **heavy metals** via fluorescence.](#metalDU)

# Engineering of the Hydro Guardian Biosensor

---

The Hydro Guardian Biosensor integrates dual sensing of antibiotic and metal residues by combining prokaryotic and eukaryotic components in two subunits, as described in the following sections. To engineer the Hydro Guardian, we employed the synthetic biology engineering cycle, encompassing the key phases: Design, Build, Test, and Learn. We iteratively applied this cycle to both subunits of our system, as outlined below.
In addition, we extended the application of the engineering cycle to experiments that supported the Design and Learn phases. For example, [Spectroscopy](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27spectroscopy%27%29%20%7D%7D), detailed separately, provided critical insights into the limitations, challenges, and requirements of our biosensor. Also, our sophisticated [Model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) further enhanced our analysis of metal and antibiotic residues through coupled differential equations and [stakeholder interviews](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) provided additional insights that reinforced our approach in the Learn and Design phases.
Below, we detail how our laboratory work on the Hydro Guardian remained centered on the iterative engineering cycle, refining each stage progressively. While some preliminary results are already mentioned here, the full dataset and findings can be found on the dedicated [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/5317/images/engineering/igem-eng-cycle.png)

Figure 1: The iGEM Engineering Cycle

## β-Lactam Detection Unit

---

To successfully implement our β-lactam detection unit, extensive literature research was conducted at the outset of the project to explore potential detection and signalling mechanisms. This phase was further supported by insights gained from spectroscopy and stakeholder interviews. Our goal was to achieve detection sensitivity at least comparable to that of spectroscopy and to exceed the legal concentration thresholds for β-lactam detection.
As outlined in the description, both detection units are intended to function together in HEK cells. Therefore, a significant challenge emerged: there is no known mammalian or eukaryotic β-lactam detection pathway. This led to our primary engineering question: Can we leverage the prokaryotic PASTA domain, coupled with downstream prokaryotic or known eukaryotic targets, to function within eukaryotic cells? We will answer this in the following. For plasmid design, we utilized SnapGene software, generously provided by SnapGene, which streamlined the process and facilitated the development of our constructs.

### Cycle 1

### Design

In the first phase, we focused on selecting the genes for four proteins that could play a role in the detection and signal transduction of beta-lactam antibiotics: *pknB, graR, ccpA* and *atf2*. Our β-lactam detection unit is based on a system that combines both bacterial and human components to detect β-lactam antibiotics in HEK cells. The function of our system is based on signal transduction, where the antibiotic acts as an external signal and triggers a signalling cascade leading to a measurable fluorescent response (Fig. 3). The central component is PknB ([BBa\_K5317013](https://parts.igem.org/Part%3ABBa_K5317013)), a serine/threonine protein kinase from *Staphylococcus aureus*, which specifically recognizes β-lactam antibiotics via its PASTA domains. This domain is characterized by its ability to recognize β-lactam compounds, which activates downstream signalling components through autophosphorylation.Text
These signalling components include the transcription factors GraR ([BBa\_K5317015](https://parts.igem.org/Part%3ABBa_K5317015)), CcpA ([BBa\_K5317014](https://parts.igem.org/Part%3ABBa_K5317014)) and ATF2 ([BBa\_K5317016](https://parts.igem.org/Part%3ABBa_K5317016)). All can be activated by phosphorylation by PknB and play a critical role in signal transduction. GraR and CcpA are also derived from *S. aureus* and are normally responsible for the activation of genes involved in bacterial adaptation to stress.TextText ATF2 is a human transcriptional activator that regulates the transcription of various genes. Particularly noteworthy is its ability to be phosphorylated by the bacterial kinase protein PknB, which activates it and influences the transcription of specific target genes.Text
All genes were codon-optimized for expression in HEK cells and fused with fluorescent reporter proteins (mRuby2 ([BBa\_K5317001](https://parts.igem.org/Part%3ABBa_K5317001)) or EGFP ([BBa\_K3338006](https://parts.igem.org/Part%3ABBa_K3338006))) to monitor their localization and activity in the cells.

### Build

After design, we introduced the genes *pknB, graR, ccpA* and *atf2* into the HEK cells by cloning them into expression plasmids under the control of a CMV promoter. These plasmids were expressed in the cells and the fluorescent labelled proteins were then examined for their localization and expression.

**Click here to look at the plasmids!** (Fig. 2)

### Test

In the first test phase, we examined the function and localization of the proteins in the cells using fluorescence microscopy. We found that only three of the four proteins showed activity and were localized; ATF2 in particular was characterized by strong expression and precise localization in the cell nucleus, while GraR shows a weak and CcpA no expression. Particularly noteworthy here is the very nice localization of the prokaryotic membrane protein PknB in the eukaryotic cell membrane. These results indicated that ATF2 was the most effective transcription factor for our detection unit. We have outlined this in much detail in the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section.

### Learn

The qualitative validation via microscopy showed that GraR and CcpA are not the suitable parts for our biosensor. Both parts were either not expressed or only expressed with a weak signal. Moreover, we learned that ATF2 and PknB showed the best performances, both in terms of its expression and its localization in the cell. With this knowledge, we decided to focus the next cycle of the engineering cycle specifically on ATF2.

![](https://static.igem.wiki/teams/5317/images/engineering/autophosphoryzation.png)

Figure 3: Autophosphorylation of PknB by recognition of β-lactam antibiotics by the PASTA domain, leading to activation of the downstream signalling cascade.

### Cycle 2

### Design

In the design process of the next cycle, we focused on the development of a new promoter specifically optimized for activation by ATF2. Based on this protein, we developed a system in which antibiotic activation leads to the binding of ATF2 to the specially designed ATF2-3xCRE3xAP1 promoter ([BBa\_K5317017](https://parts.igem.org/Part%3ABBa_K5317017)) (Fig. 5).
This promoter contains specific binding sites for ATF2 and activates the expression of a fluorescent reporter protein (miRFP670) when ATF2 binds to the promoter. The binding sites include three CRE, short for cyclic AMP-Response Element, and three AP-1, short for Activator Protein-1, sequences that increase the specificity and strength of the gene regulatory response Text. Beside ATF2, CRE-sites are getting bind by CRE binding proteins (CREB), which are part in the regulation of several cellular responses Text. CREs have the following consensus sequence 5′-TGACGTCA-3′ Text. Moreover, ATF2 is able to bind to AP-1 sites with the consensus sequence 5’-TGACTCA-3’. AP-1 proteins are containing several groups of proteins and transcription factors like Fos proteins, Jun proteins or activating transcription factors Text. This specific customization is intended to develop a system that responds to antibiotic activation and induces a measurable fluorescent response. To finalize the promoter construct, a miniCMV promoter unit was used to reach a strong expression level in the HEK cells.

### Build

Once the promoter was designed, we cloned it into a plasmid carrying the miRFP670 reporter. This allowed us to measure a fluorescent response to activation by beta-lactam antibiotics.

**Click here to look at the plasmids!** (Fig. 4)

### Test

We then tested the new promoter by adding ampicillin (0 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL, 25 µg/mL, 100 µg/mL) to the cells. This led to the activation of ATF2, which in turn triggered the specially designed promoter and induced expression of the miRFP670 reporter. The fluorescent reporter showed a response, confirming the efficacy of our design. We have outlined this in much detail in the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section.

### Learn

After the second cycle, we found that our sensor responded to antibiotics, but only to small amounts. This showed us that the basic design of the detection system works, but the sensitivity needs to be optimized to detect larger antibiotic concentrations or different β-lactam compounds more reliably. From these results, we were able to identify several potential areas for improvement:

* **Promoter strength**: It is possible that the number of CRE and AP1 binding sites in the antibiotic promoter is not yet sufficient to trigger strong ATF2-mediated gene expression. By increasing the number of these binding sites, the efficiency of ATF2 activation could be improved.
* **Modification of the signalling proteins**: The efficiency of signal transduction could be increased by targeted mutations in PknB or ATF2 to enhance the phosphorylation or binding affinity of these proteins to achieve more robust activation of the reporter.

These findings will form the basis for the next cycle in the engineering process, in which we would like to further refine our detection system and significantly improve its sensitivity.

![](https://static.igem.wiki/teams/5317/images/engineering/promoterab.png)

Figure 5: Antibiotic recognition promoter containing three CRE and three AP-1 sequences for expression of the fluorescent reporter protein miRFP670. When ATF2 is phosphorylated by PknB, it can bind to the consensus sequences of CRE and AP-1. This leads in the downstream expression of the reporter gene of miRFP670.

## Metal Detection Unit

---

Next to the successful implementation of our β-lactam detection unit, we aimed to engineer a metal detection unit applying the same challenges (e.g. minimal concentration) as above. Here, we could use a system which is already characterized to some extend in mammals and based on the Metal-responsive Transcription Factor-1 (MTF-1) as we explain in detail in the description. However, it is not yet clear if this system can be applied for metal residue detection and our following engineering cycle addressed this aim.

### Design

##### The Detector/Regulator – Metal-responsive Transcription Factor-1 (MTF-1)

The first important part is the transcription factor MTF-1 ([BBa\_K5317007](https://parts.igem.org/Part%3ABBa_K5317007)). MTF-1, short for Metal-responsive Transcription Factor-1, is found in humans, but also in mice TextText. In animal cells, MTF-1 serves as part of a defense mechanism against heavy metals, which enter the cell. Divalent ions such as cadmium, zinc or copper are recognized and bound Text (Fig. 7). Recognition of the metal ions leads to translocation from the cytoplasm into the cell nucleus. There it binds to the consensus sequence 5'-TGCRCNC-3' (R, purine; N, any nucleotide) of the MREs (metal-responsive element) in the promoter of the target gene, such as that of the metalthioneins (MT) Text. We obtained the sequence for the MTF-1 about the open source “Alliance of Genome Resources”. The sequence for mRuby2 was obtained from the pLenti Lifeact-mRuby2 PuroR plasmid.

##### The Reporter – Metal-Responsive Element (MRE) - containing Promoter

The second important part for our biosensor is the before mentioned MRE, in front of the MTs. These are small metal-binding stress proteins that play an important role in metal homeostasis and much more interestingly in detoxifying the cell from toxic metal ions Text. The wild-type (WT) promoter contains five different consensus sequences (a to e). Of these, sequences “a” (5'-CTTTGCGCCCGGACT-3') and “d” (5'-CTCTGCACTCCGCCCGA-3') are bound by MTF-1 Text. For the design of the MRE promoter, we followed the work of Searle et al. (1985)[11] and adapted the sequence order and composition accordingly. We used the following MREs for the promoter - “a” (5'-CTTTGCGCCCGGACT-3') and “d” (5'-CTCTGCACTCCGCCCGA-3') as well as the WT promoter ([BBa\_K5317003](https://parts.igem.org/Part%3ABBa_K5317003)) with the MREs “a”, “b” (5'-CTGCTGGGTGCAAAC-3'), “c” (5'-AAAGTGCGCTCGGCTC-3') and “d” Text. In addition to the WT sequence, the following arrangements were chosen for the other three promoters - 4x “a” ([BBa\_K5317004](https://parts.igem.org/Part%3ABBa_K5317004)), 4x “d” ([BBa\_K5317005](https://parts.igem.org/Part%3ABBa_K5317005)) and “dada” ([BBa\_K5317006](https://parts.igem.org/Part%3ABBa_K5317006)) (see Figure 8). To show the activity and cell localization of our components, we decided to label the MTF-1 with mRuby2 ([BBa\_K5317001](https://parts.igem.org/Part%3ABBa_K5317001)) and the respective promoters with eGFP ([BBa\_K3338006](https://parts.igem.org/Part%3ABBa_K3338006), iGEM Hannover 2020).

### Build

Based on this, we developed our metal biosensor and designed the corresponding sequences. For cloning, the promoters were amplified by PCR with the designed primers HGm\_MRE-prom\_fw and HGm\_MRE-prom\_rev with overhangs. The MTF-1 and the corresponding mRuby2 were ordered as ready-to-use GeneBlocks with matching overhangs from IDT. With the help of HiFi DNA Assembly, the respective parts were integrated into the linearized backbone vector EGFP-C2 ([BBa\_K3338020](https://parts.igem.org/Part%3ABBa_K3338020), iGEM Hannover 2020), making it possible to recreate the desired plasmids in a simple and reliable way. This resulted in the following composite parts, shown in figure 6: ([BBa\_K5317012](https://parts.igem.org/Part%3ABBa_K5317012)) & ([BBa\_K5317008](https://parts.igem.org/Part%3ABBa_K5317008), [BBa\_K5317009](https://parts.igem.org/Part%3ABBa_K5317009), [BBa\_K5317010](https://parts.igem.org/Part%3ABBa_K5317010), [BBa\_K5317011](https://parts.igem.org/Part%3ABBa_K5317011))

**Click here to look at the plasmids!** (Fig. 6)

### Test

The finished plasmids were validated and confirmed by colony PCRs and sequencing. Subsequently, the HEK293T cells were transfected with the constructs using the GenJet™ reagent. The successful transfection was confirmed the next day by fluorescence microscopy and localization in the cell was checked.
After successful confirmation of localization and basal activity of the constructs, stimulation with copper sulfate (CuSO4) could be performed. Different concentrations (0 µM, 50 µM, 100 µM, 200 µM, 300 µM & 500 µM) were tested. The different concentration levels were used to test the sensitivity of the promoter (Fig. 8). If Cu2+ ions are present in the medium, MTF-1 is activated and the MRE sequences of the synthetic promoter are bound. This leads to the expression of the reporter gene for the fluorescent protein EGFP (Fig. 9). With increasing concentration of Cu2+ ions, fluorescence should increase in comparison to the basal level.

The tests indicated the best activity for the MRE-containing promoter “dada”. There was the highest increase in fluorescent signal between basal level to stimulation (500 µM) detectable. We have outlined this in much detail in the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) section.

### Learn

What we have learned through the testing phase was that the promoter “dada” has the strongest activity. Sadly, there was no time left to improve this promoter. For the future, this promoter could be extended/shortened by “a” and “d” sequences. Moreover, the impact of the consensus sequences “b” and “c” on the synthesized promoter could be considered. Moreover, the functionality of the biosensor could and should be tested for their sensitivity in the presence of other heavy metals like zinc, cadmium or the more toxic lead.

![](https://static.igem.wiki/teams/5317/images/engineering/activationtranscription.png)

Figure 7: Activation of the Metal-responsive Transcription Factor-1 by Cu2+ ions.

![](https://static.igem.wiki/teams/5317/images/engineering/promoterhm.png)

Figure 8: MRE-containing promoter. The promoter is constructed with different combinations of MRE sequences (wildtype, 4x “a”, 4x “d” and “dada”) to which EGFP is fused. When copper or a similar heavy metal like zinc or cadmium binds to MTF-1, it gets activated. The activation leads to binding of the MRE consensus sequences followed by the expression of the reporter gene of EGFP.

![](https://static.igem.wiki/teams/5317/images/engineering/interaction.png)

Figure 9: Interaction of MTF-1 with the promoter. In a), no EGFP is expressed because no Cu2+ ions are present. After activation of MTF-1 by Cu2+ ions, it binds to the promoter, causing EGFP to be expressed.

## References

* [1] Rakette S, Donat S, Ohlsen K, Stehle T. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB. PLoS One. 2012;7(6):e39136. doi: 10.1371/journal.pone.0039136
* [2] Fridman M, Williams GD, Muzamal U, Hunter H, Siu KW, Golemi-Kotra D. Two unique phosphorylation-driven signaling pathways crosstalk in Staphylococcus aureus to modulate the cell-wall charge: Stk1/Stp1 meets GraSR. Biochemistry. 2013 Nov 12;52(45):7975-86. doi: 10.1021/bi401177n
* [3] Leiba J, Hartmann T, Cluzel ME, Cohen-Gonsaud M, Delolme F, Bischoff M, Molle V. A novel mode of regulation of the Staphylococcus aureus catabolite control protein A (CcpA) mediated by Stk1 protein phosphorylation. J Biol Chem. 2012 Dec 21;287(52):43607-19. doi: 10.1074/jbc.M112.418913
* [4] Miller, M., Donat, S., Rakette, S., Stehle, T., Kouwen, T.R., Diks, S.H., Dreisbach, A., Reilman, E., Gronau, K., Becher, D., Peppelenbosch, M.P., van Dijl, J.M., & Ohlsen, K. (2010). Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases. PLoS One, 5, (2), e9057. doi: 10.1371/journal.pone.0009057
* [5] Thiel, G., & Rössler, O. G. (2016). Resveratrol stimulates cyclic AMP response element mediated gene transcription. *Molecular nutrition & food research*, *60*, 256-265.
* [6] Andrisani, O. M. (1999). CREB-mediated transcriptional control. *Critical Reviews™ in Eukaryotic Gene Expression*, *9*.
* [7] Kim, E., Ahuja, A., Kim, M. Y., & Cho, J. Y. (2021). DNA or protein methylation-dependent regulation of activator protein-1 function. *Cells*, *10*, 461.
* [8] Greenshpan, Y., Sharabi, O., Yegodayev, K. M., Novoplansky, O., Elkabets, M., Gazit, R., & Porgador, A. (2022). The Contribution of the Minimal Promoter Element to the Activity of Synthetic Promoters Mediating CAR Expression in the Tumor Microenvironment. *International Journal of Molecular Sciences*, *23*, 7431.
* [9] LaRochelle, O., Labbé, S., Harrisson, J. F., Simard, C., Tremblay, V., St-Gelais, G., Govindan, M. V., & Seguin, C. (2008). Nuclear factor-1 and metal transcription factor-1 synergistically activate the mouse metallothionein-1 gene in response to metal ions. *Journal of Biological Chemistry*, *283*, 8190-8201.
* [10] Saydam, N., Georgiev, O., Nakano, M. Y., Greber, U. F., & Schaffner, W. (2001). Nucleo-cytoplasmic trafficking of metal-regulatory transcription factor 1 is regulated by diverse stress signals. *Journal of Biological Chemistry*, *276*, 25487-25495.
* [11] Searle, P. F., Stuart, G. W., & Palmiter, R. D. (1985). Building a metal-responsive promoter with synthetic regulatory elements. *Molecular and Cellular Biology*, *5*, 1480-1489.

## References

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* Item 4

#### Plasmid Sequence Maps

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/cmv-egfp-pknb.png)

A) CMV-EGFP-PknB ([BBa\_K5317018](https://parts.igem.org/Part%3ABBa_K5317018))

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/cmv-ccpa-mruby2.png)

B) CMV-ccpA-mRuby2 ([BBa\_K5317019](https://parts.igem.org/Part%3ABBa_K5317019))

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/cmv-grar-mruby2.png)

C) CMV-GraR-mRuby2 ([BBa\_K5317020](https://parts.igem.org/Part%3ABBa_K5317020))

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/cmv-atf2-mruby2.png)

D) CMV-ATF2-mRuby2 ([BBa\_K5317021](https://parts.igem.org/Part%3ABBa_K5317021))

Figure 2: Sequence map of the composite parts.
All four parts are under the control of a transient CMV promoter. PknB is labelled with eGPF, while the Transcription Factors ATF2, CcpA and GraR are labelled with mRuby. This allows to distinguish the different parts after co-transfection in HEK293T cells.

#### Plasmid Sequence Map

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/atf2-promoter-mirfp670-minicmv-mirfp670.png)

Figure 4: Sequence map of the AP1 (Activator Protein 1) and CRE (cAMP Response Element) Containing Promoter. ATF2-3xCre3xAP1-Promoter\_miniCMV\_miRFP670 ([BBa\_K5317022](https://parts.igem.org/Part%3ABBa_K5317022)). The promoter is labelled with miRFP670 to distinguish its fluorescent signal from the ATF2 and PknB signals.

#### Plasmid Sequence Map

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/cmv-mtf1-mruby2.png)

A) Finished construct for the CMV-MTF-1-mRuby2 plasmid.

![](https://static.igem.wiki/teams/5317/images/engineering/promoterschaerfer/mrewt-egfp.png)

B) Finished promoter construct (wildtype) with eGFP.

Figure 4: Composite parts ([BBa\_K5317012](https://parts.igem.org/Part%3ABBa_K5317012) & [BBa\_K5317008](https://parts.igem.org/Part%3ABBa_K5317008), [BBa\_K5317009](https://parts.igem.org/Part%3ABBa_K5317009), [BBa\_K5317010](https://parts.igem.org/Part%3ABBa_K5317010), [BBa\_K5317011](https://parts.igem.org/Part%3ABBa_K5317011)).
MTF-1-mRuby2 is under the control of a transient CMV promoter, while the MRE-containing promoters are activated by MTF-1. To distinguish both constructs after co-transfection in HEK293T cells, they were labeled with mRuby2 (MTF-1) or eGFP (promoters).

[Back to the Top](#engineering_BackToTop)

{% endblock %}

{% extends "layout\_experiments.html" %}
{% block title %}Experiments: Bringing Our Project to Life{% endblock %}
{% block lead %}Welcome to the Experiments page of the Hydro Guardian. Explore the laboratory work and protocols that form the backbone of our synthetic biology project and see which hands-on experiments have advanced our understanding and driven our innovations.{% endblock %}
{% block page\_content %}

Here you find our biological experiments. If you want to have a look at our additional physical experiments, check out our [Spectroscopy Analysis](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27spectroscopy%27%29%20%7D%7D).

# Chemicals and Consumables

---

## Chemicals

*Table 1: Overview over the used chemicals in our experiments*

| Chemical | Manufacturer |
| --- | --- |
| MilliQ water Q-Pod® | Merck |
| DpnI | Jena Bioscience |
| BamHI | Jena Bioscience |
| SalI | Jena Bioscience |
| NheI | Jena Bioscience |
| AseI | NEB |
| 10X rCutsmart Buffer | NEB |
| 10X Universal Buffer | Jena Bioscience |
| NEB HiFi Assembly Master Mix | NEB |
| NEB positive control reaction mix | NEB |
| Agar | Fluka Chemie AG |
| LB (Luria/Miller) – Broth | Carl Roth |
| Kanamycin (50 mg/mL) | Carl Roth |
| Ampicillin (100 mg/mL) | Carl Roth |
| SOC Outgrowth Medium | NEB |
| 10X GC Buffer | NEB |
| 10mM dNTPs | PCR Biosystems |
| DMSO | NEB |
| Phusion-Polymerase | kind gift by Prof. Dr. Jan Faix |
| 96 – 100% Ethanol | Carl Roth |
| Glycerol | Sigma-Aldrich |
| DMEM, high glucose | Sigma-Aldrich |
| FCS | Sigma-Aldrich |
| Pen/Strep (Penicillin and Streptomycin) | Sigma-Aldrich |
| 0.25% Trypsin/EDTA | Gibco |
| DPBS | Sigma-Aldrich |
| GenJet™Reagent (Ver. II) | SignaGen Laboratories |
| CuSO4 | Carl Roth |
| 1M HEPES | Carl Roth |
| FACS-Buffer (DPBS + 1%FCS) | Self-made |

## Consumables

*Table 1: Overview over the used consumables in our experiments*

| Consumable | Manufacturer |
| --- | --- |
| Pipets | Eppendorf |
| Pipet Tips | Sarstedt |
| Serological Pipets | Sarstedt |
| 1.5mL Reaction Tubes | Sarstedt |
| 2mL Reaction Tubes | Sarstedt |
| PCR-Tubes | Sarstedt |
| Cryo-Tubes | Sarstedt |
| 15mL Canonical Tubes | Sarstedt |
| 50mL Canocical Tubes | Sarstedt |
| Cell Culture Plates | Sarstedt |
| Petri Dishes, 100mm | Sarstedt |
| Zyppy™ Plasmid Miniprep Kit | Zymo Research |
| ZymoPURE II Plasmid Midiprep Kit | Zymo Research |
| HEK293T cells | DSMZ |
| NEB competent *E. coli* DH5-alpha cells | NEB |

# Cloning Strategy

---

To design and build the needed constructs, we planned the following cloning strategy. For this, in the beginning, we performed Polymerase Chain Reactions (PCR) to amplify the needed parts, which are needed for the HiFi DNA Assembly.

![](https://static.igem.wiki/teams/5317/images/experiments/figure-cloningstrategy.png)

Experimental workflow of our cloning strategy

## Polymerase Chain Reaction – PCR

A PCR can serve, on the one hand, as a preparative tool for amplification of individual fragments and, on the other hand, as an analytical tool to validate the reactions performed.

*Table 3: PCR protocol. The reaction setup for a 50 µL reaction based on the “Phusion*™*High-Fidelity DNA Polymerase” protocol by NEB.*

| Component | 50 µLReaction | Final Concentration |
| --- | --- | --- |
| 10X GC Buffer | 10 µL | 1X |
| 10 mM dNTPs | 1 µL | 200 µM |
| 10 µM Forward Primer | 2.5 µL | 0.5 µM |
| 10 µM Reverse Primer | 2.5 µL | 0.5 µM |
| DMSO (optional) | (1.5 µL) | 3% |
| Phusion DNA Polymerase | 0.5 µL | 1.0 Units/50 µL PCR |
| Template DNA | 1 µL | < 250 ng |
| Nuclease-free water | to 50 µL |  |

*Table 4: Thermocycler program. Thermocycling conditions for a routine PCR according to NEB.*

| Step | Time | Temperature | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 30 sec | 98°C | 1 |
| Denaturation | 5-10 sec | 98°C | 30 |
| Annealing\* | 10-30 sec | 50-72°C | 30 |
| Extension\* | 20-30 sec/kb | 72°C | 30 |
| Final extension | 2 min | 72°C | 1 |

\*The annealing temperature and extension time depends on primer use and product length, respectively.

*Table 5: Primerlist for all used primers for standard PCR.*

| Gene/Element | Sequence 5´->3´ |
| --- | --- |
| HGm\_MRE-prom\_fw | CCGCCATGCATTAGTTATGCACACTGGCGCT |
| HGm\_MRE-prom\_rev | TGGCGACCGGTAGCGGACGCTTAGAGGACAGC |
| MTF-1\_fw | CAGAGCTGGTTTAGTGAACCGTCAGATCCGATGGGGGAACACAGTCCAGAC |
| MTF-1\_rev | gcccttagacaccatGGGTGGCAGCTGCAGG |
| mRuby2\_fw | CTGCAGCTGCCACCCatggtgtctaagggcgaagagc |
| mRuby2\_rev | ATCCCGGGCCCGCGGTACCGTCGACTGCAGcttgtacagctcgtccatccc |
| MTF-1\_solo\_fw | CAGAGCTGGTTTAGTGAACCGTCAGATCCGATGGGGGAACACAGTCCAGAC |
| MTF-1\_solo\_rev | ATCCCGGGCCCGCGGTACCGTCGACTGCAGCTAGGGTGGCAGCTGCAG |
| Atf-2\_Fragment1\_fw | TGAACCGTCAGATCCGatgaaattcaagttacatgtgaattctgccag |
| Atf-2\_Fragment1\_rev | ggatccccacttcctgagggctgtgac |
| Atf-2\_Fragment2\_fw | caggaagtggggatccaccggtcg |
| Atf-2\_Fragment2\_rev | TCAGTTATCTAGATCCGGTGcttgtacagctcgtccatccc |
| ccpA\_Fragment1\_fw | tggatccccttttgtagttcctcggtattcaattctgtgag |
| ccpA\_Fragment1\_rev | TGAACCGTCAGATCCGatgacagttactatatatgatgtagcaagagaagc |
| ccpA\_Fragment2\_fw | actacaaaaggggatccaccggtcg |
| ccpA\_Fragment2\_rev | TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc |
| graR\_Fragment1\_fw | TGAACCGTCAGATCCGatgcaaatactactagtagaagatgacaatactttgt |
| graR\_Fragment1\_rev | tggatccccttcatgagccatatatccttttcctacttttgt |
| graR\_Fragment2\_fw | ctcatgaaggggatccaccggtcg |
| graR\_Fragment2\_rev | TCAGTTATCTAGATCCGGTGttacttgtacagctcgtccatcccacc |
| pknB\_Fragment\_fw | AGCTTCGAATTCTGCAGAatgataggtaaaataataaatgaacgatataaaattgtagataagcttgg |
| pknB\_Fragment\_rev | TCAGTTATCTAGATCCGGTGttatacatcatcatagctgacttctttttcagctacag |
| eGFP-C\_fw | GTCCTGCTGGAGTTCGTG |
| eGFP-N\_fw | CAACGGGACTTTCCAAAATG |
| SV40\_rev | cctctgcataaataaaaaaaattagtcagccatgg |
| H2A+ linker\_Cla+PCR\_rev | TCCTCGCCCTTGCTCACCATggtggcgaccgg |
| CMV\_fw | CGCAAATGGGCGGTAGGCGTG |
| eGFP-C\_rev | AGCTGCAATAAACAAGTT |
| eGFP-C1-anti | GGTTCAGGGGGAGGTGTG |
| SV40-pA\_rev | CCTCTACAAATGTGGTATGG |
| ATF2\_miRFP\_BB\_rev | cgatacaccatcacccggtcg |
| TVBB\_ColE1\_Ori\_rev | ttcgccacctctgacttgag |

## Restriction Digest

Beside the amplification of our parts, the restriction digest of the backbone EGFP-C2 was performed. The digested backbone is needed to bring the parts in the whole construct together. Different restriction enzymes are used, depending on where the backbone should be cut and whether the already present EGFP is needed or should be replaced by mRuby or miRFP.

*Table 6: Restriction digest. The reaction setup for a 50 µL reaction according to NEB and Jena Bioscience.*

| Component | 50 µL Reaction | Final Concentration |
| --- | --- | --- |
| 10X rCUTSMART Buffer/10X UB Buffer | 5 µL | 1X |
| RE1 | 1 µL | 20 units/50 µL |
| RE2 | 1 µL | 20 units/50 µL |
| Backbone DNA | 1 µg |  |
| Nuclease-free water | Add to 50 µL |  |

The reaction is incubated at 37°C for one hour. To deactivate the enzyme, the mixture is incubated at 80°C for 20 minutes.

*Table 7: Restriction digest. Used enzymes to cut the backbone EGFP-C2 for HiFi DNA Assembly.*

| Plasmids | RE1 | RE2 |
| --- | --- | --- |
| PknB EGFP C2 | SalⅠ | BamHⅠ |
| GraR mRuby2 C2 | NheⅠ | BamHⅠ |
| CcpA mRuby2 C2 | NheⅠ | BamHⅠ |
| MTF-1 mRuby2 C2 | NheⅠ | BamHⅠ |
| ATF2 mRuby2 C2 | NheⅠ | BamHⅠ |
| ATF2 miRFP670 Promoter | AseⅠ | BamHⅠ |
| MRE EGFP Promotor | AseⅠ | NheⅠ |

## Dpn Ⅰ Digestion

The DpnⅠ digestion is applied after the last PCR. The enzyme DpnⅠ belongs to type Ⅱ restriction endonucleases, which cleave at the methylated recognition sequence 5'-GmATC-3' in double-stranded DNA Text. This ensures that the remaining template is destroyed, as the desired PCR product is unmethylated. The procedure is analogue to the previous restriction digest.

*Table 8: DpnⅠ Digest. Reaction setup for a 50 µL reaction according to Jena Bioscience.*

| Component | 50 µL Reaction | Final Concentration |
| --- | --- | --- |
| 10X UB Buffer | 5 µL | 1X |
| DpnⅠ | 1 µL | 20 units/50 µL |
| Backbone DNA | 1 µg |  |

The reaction is incubated at 37°C for one hour. To deactivate the enzyme, the mixture is incubated at 80°C for 20 minutes.

## HiFi DNA Assembly

Finally, the HiFi DNA Assembly, a further developed version of the Gibson assembly, which was created by Daniel G. Gibson[2], is performed. It describes a cloning method, where multiple DNA fragments are joined together in a single reaction, regardless of size of DNA fragments and their overlaps. First, the overlapping 5´ ends of the DNA fragments are digested by an exonuclease and the 3´overhangs facilitate the annealing of the fragments at their overlapping regions. After that, a DNA polymerase extends the 3´ends by filling the gaps with dNTPs and lastly, a DNA ligase seals the nicks, so that a new double-stranded DNA construct emergesTextText. The protocol is shown in the next steps.

**Insert & Vector preparation:**

1. Inserts and vectors were amplified with overhang primers and verified according to the PCR protocol or were ordered as GeneBlocks with overhangs.

**Assembly reaction:**

1. Set up the following reaction on ice:

   *Table 9: HiFi DNA Assembly. Setup protocol to assemble 1 to 2 inserts according to NEBuilder® HiFi DNA Assembly protocol by NEB.*

   | Component | 2–3 Fragment Assembly |
   | --- | --- |
   | Recommended DNA Molar Ratio | vector:insert = 1:2 |
   | Total Amount of Fragments | X µL = 100 ng vector : 200 ng inserts \* |
   | NEBuilderHiFi DNA Assembly Master Mix | 10 µL |  |
   | Deionized H2O | 10-X µL |  |
   | Total Volume | 20 µL |  |

   \*Volume of fragments depends on measured concentration.
2. Incubate samples in a thermocycler at 50°C for 1 hour. For following incubation, store samples on ice or at –20°C for subsequent transformation.
3. Transform competent *E. coli* DH5 alpha (NEB) cells with 2 μl of the chilled assembled product, following the transformation protocol (NEB).

## Heatshock Transformation

The ready to use plasmids are transferred in Competent *E. coli* DH5-alpha cells via heatshock transformation. The plating of the transformed cells on LB-Agar plates containing the antibiotic Kanamycin ensures that just positive cells, containing the plasmid with the Kanamycin resistance cassette, can grow.

**Agar plate preparation:**
Three plates of Kanamycin (50 µg/mL) containing LB-Agar plates (1.5% Agar) were prepared before starting the transformation protocol. For this, approximately 15 mL is used per plate. Two plates are for plating the bacteria, one is used as negative control and subsequent master plate.

**Protocol:**
Competent NEB®5-alpha *E. coli* bacteria, which were kindly sponsored by New England Biolabs, were used according to manufacturer´s protocol to introduce the cloned plasmids into bacteria for propagation of the expression vectors. For this purpose, 50 µL of NEB®5-alpha *E. coli* was mixed with 2 µL of the respective vector and incubated on ice for 30 minutes. Thereafter, heat shock was performed at 42°C for exactly 45 seconds. For recovery, the bacteria were placed on ice for a few minutes and 250 µL of SOC Outgrowth Medium (room temperature) was added. In this medium, the bacteria were shaken at 37°C and 180 rpm for one hour and then spread on agar plates containing kanamycin (50 µg/mL). Overnight, the bacteria incubated at 37°C and were selected by the antibiotic.

## Colony PCR

By picking the grown colonies from the agar plates, colony PCRs can be performed. Via this method, the correctness of the inserts can be validated and if the clones can be used for the next steps. For the colony PCR are different primers used than for the amplification.

**Colony-PCR each with 20 µl preparations with for 10 clones each:**

1. Colony PCR and master plate preparation.
   * Prepare 10x 1.5 mL reaction tubes for each cloning approach by adding 20µl ddH2O into each tube.
   * Pick a nicely grown clone without visible satellite colonies using a sterile pipette tip, transfer a small amount of bacterial colony to master plate by tipping onto prepared and sufficiently labeled agar plate, and place tip – still containing bacteria – into ddH2O within prepared 1.5 mL tube.
   * Repeat previous step 9x, so that there will be ten clones to be analyzed in total.
   * Transfer 5µl ddH2O including bacteria into a PCR reaction tube for each clone.
   * Incubate PCR reaction tubes for 10 min at 80 °C. This step causes lysis of the dissolved bacteria, thereby releasing potential plasmid DNA from within the cells and facilitating the use of this preparation as template for the following analytical PCR reaction.
2. Prepare Master-mix.

   *Table 10: Colony PCR. The reaction setup for a 12.5 µL and 20 µL reaction based on the “Phusion® High-Fidelity DNA Polymerase” protocol by NEB.*

   | Component | Volume [µL] (other constructs) | Volume [µL] (PknB) |
   | --- | --- | --- |
   | 10X GC-Puffer | 2.5 | 4 |
   | 10 mM dNTPs | 0.25 | 0.4 |
   | 10µM Forward Primer | 0.625 | 1 |
   | 10 µM Reverse Primer | 0.625 | 1 |
   | Phusion-Polymerase | 0.125 | 0.2 |
   | Template | 5 | 5 |
   | DMSO (optional) | 0.375 | 0.6 |
   | ddH2O | Fill up to 12.5 | Fill up to 20 |
   | **Total volume** | **12.5** | **20** |
3. Run the respective PCR program in the thermal cycler.

   *Table 11: Thermocycler program. Thermocycling conditions for a routine PCR according to NEB.*

   | Step | Time | Temperature | Cycles |
   | --- | --- | --- | --- |
   | Initial denaturation | 30 sec | 98°C | 1 |
   | Denaturation | 5-10 sec | 98°C | 30 |
   | Annealing | 10-30 sec | 50-72°C | 30 |
   | Extension | 20-30 sec/kb | 72°C | 30 |
   | Final extension | 2 min | 72°C | 1 |

   The annealing temperature and extension time depends on primer use and product length, respectively.

   *Table 12: Conditions for Colony PCR. M1.1 to M1.4 and M3.0 for metal constructs. A1 to A5 for antibiotic constructs.*

   | Construct/Vector | Annealing Temp | Elongation time | Used for primer | Used rev Primer | DMSO |
   | --- | --- | --- | --- | --- | --- |
   | M1.1 | 72°C | 15 sec | HMG-MRE-Promotor\_fw | H2a+linker Cla+PCR\_rev | + |
   | M1.2 | 72°C | 15 sec | HMG-MRE-Promotor\_fw | H2a+linker Cla+PCR\_rev | + |
   | M1.3 | 72°C | 15 sec | HMG-MRE-Promotor\_fw | H2a+linker Cla+PCR\_rev | + |
   | M1.4 | 72°C | 15 sec | HMG-MRE-Promotor\_fw | H2a+linker Cla+PCR\_rev | + |
   | M3.0 | 56°C | 1 min | eGFP-N\_fw | eGFP-C\_rev | - |
   | A1 | 65°C | 30 sec | eGFP-N\_fw | eGFP-C\_rev | - |
   | A2 | 65°C | 30 sec | eGFP-N\_fw | eGFP-C\_rev | - |
   | A3 | 63°C | 1 min | eGFP-C\_fw | eGFP-C\_anti | + |
   | A4 | 68°C | 1 min | CMV\_fw | SV40\_rev | - |
   | A5 | 65°C | 30 sec | ATF2\_miRFP\_BB\_rev | TVBB\_ColE1\_Ori\_rev | - |

## Plasmid Preparation

After the validation of the correct insert via colony PCR, the plasmid preparation was performed. The Mini and Midi Preps are necessary to extract and purify the plasmids. Finally, the plasmids are sequenced by Microsynth SeqLab to validate the purified DNA sequences so there are no mutations introduced through the replication.

**Overnight Culture:**

1. 5 ml LB
2. Add 5 µl of kanamycin from a 50 mg/mL stock solution to achieve a final concentration of 50 µg/mL.
3. Use the remaining 15 µL of the previously prepared solution of the picked bacterial clone within ddH2O (see section colony PCR) to inoculate the overnight culture.
   Incubate the culture at 37 °C with shaking at 180 rpm overnight.

**Glycerol Stock:**

1. Prepare a 50% bacterial glycerol stock solution by mixing 500 µL of 100% glycerol with 500 µL of the overnight bacterial culture.
2. Store the glycerol stock at -80 °C.

**Plasmid Preps:**
All plasmid preps were conducted using Mini and Midi Prep Kits, generously sponsored by Zymo Research, according to the manufacturer’s protocol.

For the Plasmid Midi Prep a 5 mL LB + Kan pre-culture was prepared (according to the overnight culture section) and is incubated for 3-4 hours. 1 mL from the pre-culture was used to inoculate 150 mL of fresh LB medium + 150 µL kanamycin and was incubated overnight at. The next day, 1:1000 chloramphenicol was added to the overnight culture and incubated for another one hour before prepping.

**Plasmid Validation via Sequencing:**
Use 1 µg of plasmid DNA after elution and add nuclease-free water to 12 µL total volume.

# HEK293T Cells as a Model System

---

The correct validated and purified plasmids are now ready be to introduced them in the HEK293 cells enable sensing for with CuSO4 and Ampicillin. To perform the stimulation experiments the following cell culture strategy is introduced.

First of all, a functional running culture of HEK293T cells should be introduced. The cells are regularly seeded and split according to the following protocol. This enables us to estimate and plan the right number of cells for the transfection experiments.

## Splitting of HEK293T Cells

For running culture (on Monday & Friday) and transfection culture (18 to 24 hours before transfection):

1. Pre-heat DPBS and DMEM with 10% FCS and 1% Pen/Strep or without Pen/Strep in water bath at 37°C.
2. Remove media from cells.
3. Wash cells with 5 mL DPBS.
4. Detach cells with 0.25% Trypsin+EDTA (2mL) and incubate for 5 min at 37°C.
5. Add double volume of medium (4mL) to stop reaction.
6. Centrifuge at 500 rcf for 5 min.
7. Discard supernatant and resuspend pellet in 1 mL medium.
8. Cell counting:
   1. Dilute cells 1:50.
   2. Prepare Neubauer chamber and add approx. 10 µL of diluted cells.
   3. Count the 4x4 fields four times and calculated mean value.
9. Formulas to calculate your cell number and number of cells to seed:

   *Equation 1: Determination of the cell count per mL*

   ![](https://static.igem.wiki/teams/5317/equations/experiments/eq1.png)

   *Equation 2: Determination of the cell suspension volume for transfection or the running culture*

   ![](https://static.igem.wiki/teams/5317/equations/experiments/eq2.png)
10. Use for running culture 100mm Petri Dish or 24/48 Well plate.
    1. Use 10mL of medium and transfer calculated volume of cell suspension.
    2. Use 300µL for 48 Well plates or 500µL for 24 Well plates and transfer calculated volume of cell suspension.
11. Incubate at 37°C with 5% CO2.

## Transfection of HEK293T Cells

Transfection is used to insert and express foreign DNA, like our designed plasmids, into eukaryotic cellsText. For this, we use GenJet™ In Vitro DNA Transfection Reagent (Ver. II). A transfection efficiency of about 80 to 90% should be reached in HEK-293T cellsText. The transfection is proceeded by following the protocol of SignaGen® Laboratories.

*Table 13: The transfection setup for transfectiong cells in 6 Well, 24 Well and 48 Well Culture Dishes based on the “GenJet™ In Vitro DNA Transfection Reagent (Ver. II)” protocol by SignaGen*®*Laboratories.*

| Culture Dish | Culture Medium [mL] | Plasmid DNA [µg] | Diluent Volume [mL] | GenJet™ Reagent [µL] |
| --- | --- | --- | --- | --- |
| 48 Well Plate | 0.3 | 0.25 | 2 x 0.015 | 0.75 |
| 24 Well Plate | 0.5 | 0.5 | 2 x 0.025 | 1.5 |
| 6 Well Plate | 2 | 1 | 2 x 0.05 | 3 |

* **Part 1 – Cell Seeding:**
  1. Plate the cells 18 to 24 hours before transfection.
  2. A confluency of 70 to 80% is recommended.
  3. Change medium 30 to 60 minutes before transfection (Use DMEM High Glucose with 10% FCS and 1% Pen/Strep or without Pen/Strep).
* **Part 2 – Preparation of GenJet********-DNA Complex and Transfection Procedures:**
  1. The recommended GenJet™ (µL)-DNA (µg) ratio of 3:1 (Table 13) is used.
  2. Use serum-free DMEM with High Glucose with 1% Pen/Strep or without Pen/Strep to dilute the GenJet Reagent and the plasmid DNA.

     The following steps depending on culture dish size (Table 13):
  3. Add X mL fresh DMEM with 10% FCS and 1% Pen/Strep or without Pen/Strep 30-60 min before transfection.
  4. Dilute X µg DNA to X mL serum-free DMEM with High Glucose with 1% Pen/Strep or without Pen/Strep; diluent volume -> mix by gently vortexing.
  5. Dilute X µL GenJet™ Reagent into X mL serum-free DMEM with High Glucose with 1% Pen/Strep or without Pen/Strep; diluent volume -> mix by pipetting 3-4 times.
  6. Transfer diluted GenJet™ Reagent to diluted DNA and mix by pipetting 3-4 times and incubate for approx. 10 mins at room temperature to form the GenJet™ -DNA Complex.
  7. Add the GenJet™-DNA Complex drop wise onto the media in each well and swirl gently.
  8. Change medium after 12 to 18 hours.
  Note: for transfection with two constructs divide the total amount of DNA and use equal amounts of each plasmid DNA (µg) (Table 13).
  -> volume of GenJet™ Reagent does not change

## Stimulation via Antibiotics and Metals

After successful transfection, the HEK293T cells should be able to indicate the presence of copper sulphate and ampicilin. Different concentrations were used to stimulate the expression of the fluorescent proteins and thus the detection of metals and antibiotics. The stimulation protocol is shown below.

**CuSO****4****:**
The metal solutions were added after 24 h after of transfection. Before stimulation, the transfected cells were imaged to check the basal fluorescence signal. Moreover, 10 mM HEPES was added to fresh medium. The bivalent ions getting in the cells and bind to the MTF-1, which induces the MRE-containing promoters, which leads to eGFP expression. The maximal concentration of 2 mM, for CuSO4, is according to WHO guidelines.

*Table 14: Copper stimulation. Used concentrations for viability test and induction.*

| Concentration viability test [µM] | Concentration for induction [µM] |
| --- | --- |
| 2000 | 500 |
| 1000 | 300 |
| 500 | 250 |
| 250 | 200 |
| 50 | 100 |
| 0 | 50 |
|  | 0 |

**Ampicillin:**
Ampicillin was added after 24 h after transfection. Before the stimulation, the transfected cells were imaged to check the basal fluorescence signal before and after. The PASTA domain of PknB recognize beta-lactams, leading to phosphorylation of ATF2, which then binds to the AP1 and CRE sites in the promoter. The binding leads to the expression of miRFP670.

*Table 15: Ampicillin stimulation. Used concentrations for viability test and induction.*

| Concentration viability test [µg/mL] | Concentration for induction [µg/mL] |
| --- | --- |
| 500 | 100 |
| 100 | 25 |
| 50 | 10 |
| 25 | 5 |
| 5 | 2.5 |
| 0 | 0 |

Before stimulation and after an incubation time of 4 hours, the cells were imaged by confocal microscopy. By this, we can qualitatively validate the reporter gene expression and localization of our membrane protein (PknB) and transcription factors.

## Imaging

To check the localization and expression of the proteins, confocal microscopy is used. Confocal microscopy is an advanced optical imaging technique that enhances the resolution and contrast of micrographs by using a spatial pinhole to block out-of-focus light. Unlike conventional wide-field microscopy, which captures light from all focal planes, confocal microscopy focuses on a single plane at a time, producing high-resolution, three-dimensional images. This is achieved by scanning the specimen point by point with a laser and collecting the emitted light through a pinhole that aligns with the focus plane. The result is sharp images with minimal background noise, making confocal microscopy ideal for detailed studies of cells, tissues, and other biological specimens. The images were recorded with a 25x/0.9 water-immersion objective. For the detection of the fluorescent signal, lasers with wavelengths of 488nm, 543nm & 630nm were used. The pictures were recorded with a frame average of four and a format of 1014 x 1024 pixel.

Later, the pictures were taken with a Leica camera system. For this, channels for eGFP (488 nm), mRuby (543 nm) and miRFP670 (630 nm) were chosen to validate the fluorescent signal. For brightfield recordings exposure times of 1 ms are used, for eGFP up to 70 ms and for mRuby between 100 ms and 1000 ms depending on signal strenght.

For both setups a pinhole of 58 µM and 75 µM was used to adjust the brightness. In general, all setup points depending on the sample and the fluorescent signal.

The pictures were analyzed via the Fiji (ImageJ) software.

## FACS (Fluorescent Activated Cell Sorting) Flow Cytometry

Beside the qualitative validation of the gene expression, we want to quantify the expression level of the promoter coupled reporter genes. FACS analysis is an appropriate method to verify the functionality of the bio sensors.

Fluorescent Activated Cell Sorting (FACS) is a specialized type of flow cytometry that allows sorting and analysis of cells based on specific characteristics. Cells are first labeled with eGFP (metal promoter) or miRFP670 (antibiotic promoter). As the cells pass through a laser beam in a fluid stream, they emit light at different wavelengths depending on the markers present. In our experiments we use lasers with wavelengths of 488 nm (blue) and 635 nm (red). Detectors capture this light, and the cells are then sorted into different containers based on the fluorescent signals they emit. We use this technique to quantify our promoter activities with and without metal or antibiotic induction.

Protocol:

1. Discard media and wash with 300 µL DPBS.
   * Discard DPBS.
2. Add 150 µL 0.25% Trypsin-EDTA.
   * Incubate at 37°C for 5 min.
3. Stop reaction with 300 µL DMEM with 10% FCS & 1% Pen/Strep and transfer volume to FACS-Tubes.
4. Centrifuge at 800 rcf for 5 min.
5. Discard flow-through.
   * Resuspend pellet in 1 mL FACS-Buffer.
   * Repeat step 4.
6. Resuspend pellet in FACS-Buffer (volume depends on pellet size and cell number).
7. FACS measurement.
8. Analysis with FCSalyzer software and Microsoft Excel.

## References

* Item 1
* Item 2
* Item 3
* Item 4

[Back to the Top](#exp_top)

{% endblock %}

{% extends "layout\_results.html" %}
{% block title %}Results: Achievements of the Hydro Guardian{% endblock %}
{% block lead %}Welcome to the Results page of the Hydro Guardian. Explore the data, findings, and insights that demonstrate the effectiveness of our innovative biosensor and the impact we've made this year.{% endblock %}
{% block page\_content %}

* ![](https://static.igem.wiki/teams/5317/logos/hg-gespiegelt.png)

  [**β-Lactam Detection Unit**: Demonstrated high sensitivity, successfully detecting **β-lactam antibiotics** at **low concentrations**, with consistent **fluorescent signals** correlating to antibiotic presence.](#ResultsBetaLactamDetectionUnit)
* ![](https://static.igem.wiki/teams/5317/logos/hg-gespiegelt.png)

  [**Metal Detection Unit**: Accurately sensed **heavy metals**, with a clear relationship between **metal concentration** and **fluorescence intensity**, validating its effectiveness in water contamination detection.](#ResultsMetalDetectionUnit)

# Results – β-Lactam Detection Unit

---

To develop our cell-based sensor for early and sensitive detection of antibiotic residues in wastewater, we designed various genetic constructs and transfected cells with three plasmids designed by us. The plasmids coding for a transmembrane protein, transcription factors and a reporter fluorophore driven by a specifically designed promoter are composed of different individual parts and were validated in several steps.

After cloning success was verified by PCR and subsequent gel electrophoresis as well as sequencing, our first set of cell culture experiments aimed for the validation of the functionality of the individual plasmids we designed. In the second step, co-transfection of multiple plasmids into HEK293T cells and subsequent analysis of reporter activity upon challenge with ampicillin was investigated.

### Cloning of the Parts

---

Prior to the transfection experiments, the sequences for PknB (BBa\_K5317013), CcpA (BBa\_K5317014), GraR (BBa\_K5317015) and ATF2 (BBa\_K5317016) were inserted into the plasmid-backbone pEGFP-C2 (BBa\_K3338020). For validation of localization and fluorophore intensity, fluorescent proteins like mRuby2 (BBa\_K5317001) and EGFP (BBa\_K3338006) were used. Via HiFi DNA Assembly, our designed composite parts were integrated into one plasmid each, which were introduced into *E. coli* DH5-alpha via heat shock transformation in the following. The correct transformation of competent bacteria was validated by colony PCR, followed by an expansion of the respective bacterial clones in liquid culture, enabling plasmid isolation and conclusive sanger sequencing. The ready-to-use composite parts for our antibiotic biosensor are shown in the following Figures 1 to 4, which present a schematic plasmid feature map as well as gel electrophoresis images of PCR amplicons obtained for cloning validation purposes.

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-1.png)

**Figure 1: Detection Unit CMV-EGFP-PknB**. In silico-designed plasmid of the composite part BBa\_K5317018 and validation of correct assembly within plasmid backbone via colony PCR analysis and sequencing.

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-2.png)

**Figure 2: Regulation Unit CMV-CcpA-mRuby2**. In silico-designed plasmid of the composite part BBa\_K5317019 and validation of correct assembly within plasmid backbone via colony PCR analysis and sequencing.

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-3.png)

**Figure 3: Regulation Unit CMV-GraR-mRuby2.** In silico-designed plasmid of the composite part BBa\_K5317020 and validation of correct assembly within plasmid backbone via colony PCR analysis and sequencing.

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-4.png)

**Figure 4: Regulation Unit CMV-ATF2-mRuby2.** In silico-designed plasmid of the Composite Part BBa\_K5317021 and validation of the correct integration within plasmid backbone via colony PCR analysis and sequencing.

The successful construction of the *in silico*-designed composite parts is illustrated in Figures 1 to 4. These include CMV-EGFP-PknB (BBa\_K5317018), CMV-CcpA-mRuby2 (BBa\_K5317019), CMV-GraR-mRuby2 (BBa\_K5317020), and CMV-ATF2-mRuby2 (BBa\_K5317021). The correct introduction of our constructs was shown via analytic PCR for the clones five and nine of BBa\_K5317018 (2229 base pairs) and for clone 10 of BBa\_K5317021 (3444 bp). The correct introduction of the constructs BBa\_K5317019 (1977 bp) and BBa\_K5317020 (1665 bp) could be shown via Colony PCR. A further validation via sequencing was conducted for all plasmids to verify no mutations were inserted into the sequence during the cloning procedure.

#### ATF-2-3xCre3xAP1-miniCMV-miRFP670 (BBa\_K5317022)

The ATF2-responsive promoter 3xCre3xAP1-miniCMV-miRFP670 was designed in the second cycle of the engineering circle. The *in silico-*constructed part 3xCre3xAP1-miniCMV promoter (BBa\_K5317017), and the part miRFP670 (BBa\_K5317002) were introduced in the backbone vector pEGFP-C2 via HiFi DNA Assembly, followed by heat shock transformation of the assembled plasmid into *E. coli* DH5-alpha.

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-5.png)

**Figure 5: Expression Unit 3xCre3xAP1-miniCMV-miRFP670**. In silico-designed plasmid of the composite part BBa\_K5317022 and validation of the correct integration within the plasmid backbone via colony PCR analysis and sequencing.

The successful construction of the composite part ATF2-3xCre3xAP1-Promoter\_miniCMV\_miRFP670 is shown in figure 5. Here, you can see that the composite part ATF2-3xCre3xAP1-Promoter\_miniCMV\_miRFP670 is replacing the CMV-promoter and EGFP sequence originally present in the plasmid backbone. The correct introduction of our construct was shown for the clone seven via colony PCR, where a band with the expected sizeof 1159 bp was detectable. A further validation via sequencing was performed to verify no mutations were introduced into the sequence.

### Fluorescence Visualization Validates Assumed Localization of Components

---

![](https://static.igem.wiki/teams/5317/images/results/fig-6.png)

**Figure 6: Representative microscopy image of HEK cells expressing EGFP-PknB.** Shown are bright field channel (left), fluorescence channel (center) and an overlay of both channels (right).

As shown in Figure 6, EGFP-PknB is correctly expressed in HEK293T cells. Very delightful is the membranous installation of the codon-optimized prokaryotic membrane protein in the eukaryotic cell membrane. The presence of the protein in the cells has been a very important and challenging part of our work, as the PASTA domain of PknB is crucial for β-lactam detection in the extracellular medium. With this, we were able to continue to find a functional transcription factor for signal transfer in the cell.

![](https://static.igem.wiki/teams/5317/images/results/fig-7.png)

**Figure 7: Representative microscopy image of HEK cell expressing ATF2-mRuby.** Shown are bright field channel (left), fluorescence channel (center) and an overlay of both channels (right).

Within the first round of transfections, we discovered no or a very weak expression of our composite parts Ccpa-mRuby2 and GraR-mRuby2. As a result of this, we decided not to use these constructs for the co-transfection with EGFP-PknB. The correct expression of ATF2-mRuby2 is shown in Figure 7, where a cytoplasmatic localization in the cells is detectable. Therefore, we decided to use ATF2 as a crucial transfection factor to mediate between the PknB and the promoter. However, it is important to note that only small sample sections are presented here, and transfection efficiency may vary between treatments.

![](https://static.igem.wiki/teams/5317/images/results/fig-8.png)

**Figure 8: Representative microscopy image of HEK cells expressing EGFP-PknB and ATF2-mRuby2.** Shown are brightfield (left), fluorescence channels for eGFP and mRuby2 (both images in the center) and an overlay of the three channels (right).

The co-transfection of the functional EGFP-PknB and ATF2-mRuby2 is shown in Figure 8. The expression of both parts was detectable, which are also located in one cell. This enables us to plan in a further step a promoter, which will be activated by ATF2.

![](https://static.igem.wiki/teams/5317/images/results/fig-9-a-b.png)

**Figure 9: Representative microscopy image of HEK cells expressing EGFP-PknB, ATF2-mRuby2 and 3xCre3xAP1-miniCMV-miRFP670.** Shown are the fluorescence channels for eGFP, mRuby2 and miRFP670 (first three images from the left) and an overlay of the three channels (right). In a) is shown the basal activity of the promoter. In b) is shown the promoter activity after induction with 100 µg/mL ampicillin after four hours of incubation.

Here, Figure 9 shows HEK cells co-transfected with our composite parts EGFP-PknB and ATF2-mRuby2 as well as our tested promoter-driven reporter. ATF2 expression is shown in red and the respective reporter miRFP670 expression, which indicated a basal promoter activity, in pink. Particularly noteworthy here is again the correct localization of the prokaryotic membrane protein PknB in the eukaryotic cell membrane, in green.  As a human transcription factor, ATF2 plays a crucial role in DNA-binding to regulate gene expression Text. A basal activity of our promoter could therefore be due to possible ATF2-activity in the HEK cells that could interact with the promoter even without further antibiotic stimulus from the environment (Figure 9a).

To study whether the presence of β-lactam antibiotics in the cell media will be sensed by PknB, leading to a phosphorylation of ATF2 and subsequently to an induction of our promoter-driven reporter fluorophore, we incubated co-transfected HEK293T cells with ampicillin (100 µg/mL) for four hours.
The change of the miRFP670 reporter’s fluorescence intensity upon challenge was microscopically analyzed and is captured in the Figure 9b. Here, a qualitative change could be shown, which indicates a functional PASTA domain activity followed by ATF2 phosphorylation leading to miRFP670 expression. To address whether this change in reporter fluorescence intensity can also be aquired in a quantitative manner, we performed a flow cytometry analysis.

### Flow Cytometry Validates Fluorescence Expression Level After Ampicillin Stimulation

---

![](https://static.igem.wiki/teams/5317/images/results/fig-10.png)

**Figure 10: Quantitive validation of reporter activity by flow cytometry analysis.** The percentage of cells expressing the fluorophore miRFP670 under the control of the tested 3xCre3xAP1-miniCMV promoter is displayed as a function of various concentrations of ampicillin.

The qualitative change in the fluorescence signal was finally determined quantitatively using FACS analysis. The results are presented in the bar chart displayed in Figure 10. Here, the percentage of cells expressing the fluorophore under the control of the tested 3xCre3xAP1-miniCMV promoter is displayed as a function of various concentrations of ampicillin. A general increase across the various concentration levels can be seen after four hours of incubation, with the greatest increase from the basal level to 2.5 µg/mL. As a result, a saturation can be recognized for higher concentrations. Therefore, it can also be said here that a successful detection of ampicillin by our sensor is possible, but a closer look at concentrations between 0 µg/mL and 2.5 µg/mL will be interesting to study in future experiments.

# Results – Metal Detection Unit

---

In addition to β-lactam antibiotic detection, we designed an additional innovative cellular sensor for the detection of heavy metal residues in wastewater, such as copper, zinc and cadmium. To do this, we developed various genetic constructs and transfected cells with two self-designed plasmids. One of the plasmids contains the Metal-responsive Transcription Factor-1 (MTF-1), the other the associated MRE-containing promoter.

MTF-1 (BBa\_K5317007), as a nucleocytoplasmic shuttle protein, plays a crucial role in the recognition and cellular response to heavy metals. When heavy metals are present in the cell, it accumulates in the cell nucleus, where it binds to promoters containing a Metal-Responsive-Element (MRE) and thus specifically regulates gene expression Text. For our MRE-containing promoters, we designed *in silico* four different basic and composite parts. We used the wildtype (BBa\_K5317003) sequence of the metallothionein promoter Text as one version and for the others 4x the sequences “a” (BBa\_K5317004), “d” (BBa\_K5317005) or the combination of both “dada” (BBa\_K5317006). As an example, we show the ready-to-use plasmid of the MREwt promoter (Figure 12).

### Cloning of the Parts

---

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-11.png)

**Figure 11: Detection/Regulation Unit CMV-MTF1-mRuby2**. In silico-designed plasmid of the composite part BBa\_K5317012 and validation of the correct integration within the plasmid backbone via colony PCR analysis and sequencing.

The successful construction of the composite part CMV-MTF1-mRuby2 (BBa\_K5317012) is shown in Figure 11. Here, you can see that CMV-MTF1-mRuby2 is replacing the eGFP sequence within the plasmid backbone. The correct assembly of our construct was shown for the clone five and eight via colony PCR, where a band with the expected size of 3019 bp was detectable. A further validation via sequencing was conducted and verified no mutations were inserted within the sequence during cloning.

#### MRE-containing Promoters

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig-12.png)

**Figure 12: Expression Unit MREwt-EGFP**. In silico-designed plasmid of the composite part BBa\_K5317008 and validation of the correct integration within the plasmid backbone via colony PCR analysis and sequencing.

The successful assembly of the composite parts MREa-EGFP (BBa\_K5317009), MREd-EGFP (BBa\_K5317010), MREdada-EGFP (BBa\_K5317011), validated via PCR analysis, are shown in Figure 12, where also a representative plasmid feature map of MREwt-EGFP (BBa\_K5317008) is displayed. As can be seen, MREwt-EGFP was integrated into the plasmid backbone replacing the originally present CMV-promoter, thereby  driving the expression of the eGFP reporter. The correct assembly of our constructs was shown for all MRE-containing promoters by PCR analysis yielding a band at 230 bp on the agarose gel. A further validation via sequencing was performed for all plasmids, which verified sequence correctness.

### Fluorescence Visualization Validates Assumed Localization of Components

---

![](https://static.igem.wiki/teams/5317/images/results/fig-13.png)

**Figure 13: Representative microscopy image of HEK cells expressing MTF1-mRuby2.** Shown are brightfield (left), fluorescence channel for mRuby2 (center) and an overlay of both channels (right).

In Figure 13, the expression of MTF1-mRuby2 in HEK293T cells is illustrated in red. The localization in the cell nucleus should be emphasized here. MTF-1 plays an important role for our metal biosensor, as it recognizes and binds heavy metals such as cadmium, zinc or copper in the cytoplasm Text. This leads to activation and binding to the promoter.

Moreover, a basal activity of the MRE-containing promoters without co-transfected MTF-1 were examined. These experiments showed no fluorescent signals, which indicates that there is no induction and that MTF-1 is probably necessary for promoter activity. With this information, we were able to continue with co-transfections of both parts.

![](https://static.igem.wiki/teams/5317/images/results/fig-14-a-d.png)

**Figure 14: Representative microscopy images of HEK cells co-transfected with MTF-1-mRuby2 with each promoter MREwt-eGFP (a), MREa-eGFP (b), MREd-eGFP (c) or MREdada-eGFP (d).** Shown are brightfield channels (left), fluorescence channels (images in the center) and an overlay of the channels (right).

In the second round, we co-transfected HEK cells with our MTF1-mRuby2 composite part and our four tested promoter-driven reporters. MTF-1 expression is shown in red and the respective reporter eGFP expression, which indicated promoter activity, in green (Figure 14a-d). We observed, that MTF-1 was continuously localized within the nucleus, while the reporter fluorophore showed a cytoplasmic localization, both as expected. A basal expression of the promoter-driven reporter fluorophore can be seen for all four promoters in case of no metal stimulation. This is due to possible metal ions in the culture medium of the HEK cells that could interact with the MTF-1. To evaluate whether the reporter fluorescence can be induced in case of the presence of metals in the cellular environment, we relied on microscopic analysis as a first qualitative approach.

![](https://static.igem.wiki/teams/5317/images/results/newfigs/fig15-neu-scale-20-m-neu-klein.webp)

**Figure 15: Representative microscopy images of HEK cells co-transfected with MTF-1-mRuby2 and either promoter-reporter construct MREwt (a,b) or MREdada (c,d)**  before (a,c) or after (b,d) stimulation with 500 µM CuSO4 for four hours.

After successfully localizing and expressing the composite parts, we conducted a further qualitative evaluation of reporter signal intensity in response to 500 µM copper sulfate. The fluorescence signals for the MREwt (Figure 15a, b) and MREdada (Figure 15c, d) promoters together with MTF-1 were observed before and after four hours of stimulation. Unfortunately, no clear visual increase in fluorescence was detected for these promoters compared to the baseline signal. However, it is important to note that only small sample sections are presented here, and transfection efficiency may vary between treatments. Visual assessment of fluorescence changes is challenging, which is why flow cell cytometry was subsequently considered to obtain a quantitative analysis.

### Flow Cytometry Validates Fluorescence Expression Level After CuSO4 Stimulation

---

![](https://static.igem.wiki/teams/5317/images/results/fig-16.png)

**Figure 16: Quantitive validation by flow cytometry analysis.** The percentage of cells expressing the fluorophore under the control of the tested promoter is displayed as a function of various concentrations of copper sulfate across all four promoters.

The change in fluorescence signal was quantitatively assessed using FACS analysis. The results are presented in the bar chart in Figure 16. The percentage of cells expressing the fluorophore under the control of the tested promoter is displayed as a function of various concentrations of copper sulfate across all four promoters. It is evident that the MREa and MREdada promoters show an increase in activity, while the MREwt promoter exhibits no significant change, and MREd even shows a decrease in promoter activity upon stimulation. Notably, the MREdada-containing promoter exhibited the highest increase in fluorescence signal. Overall, our synthetic promoters, MREa and MREdada, enable effective detection of copper sulfate compared to the wild-type promoter.

# Summary of Our Results

---

We successfully designed biosensors for detecting both metals and β-lactam antibiotics.

For antibiotic detection, we introduced PknB, ATF2, and a promoter-driven reporter into HEK cells. Although challenging, we achieved successful localization of the prokaryotic membrane protein PknB in a eukaryotic system. Using the ATF2-sensitive 3xCre3xAP1-miniCMV promoter (BBa\_K5317017), we demonstrated sensitive detection of up to 2.5 µg/mL ampicillin, both qualitatively and quantitatively. For higher concentrations, a saturation in fluorescent signal was measurable.

For metal detection, we integrated the transcription factor MTF-1 and four types of MRE-containing promoters. The MREa-EGFP and MREdada-EGFP constructs showed improved fluorescent reporter expression compared to the MREwt promoter, with detection up to 500 µM copper sulfate.

Despite these successes, fine-tuning is still needed for both detection systems, which we plan to address in future experiments.

## References

* Item 1
* Item 2
* Item 3
* Item 4

## ![](https://static.igem.wiki/teams/5317/images/team/hydroguardian-transparent-60.png)

* [Back to the Top](#results_BackToTop)

  ---
* [β-Lactam Detection Unit](#ResultsBetaLactamDetectionUnit)

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* [Cloning of the Parts](#CloningABParts)

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* [Fluorescence Visualization](#FluorescenceVisualizationAB)

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* [Flow Cytometry](#FlowCytometryAB)

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* [Metal Detection Unit](#ResultsMetalDetectionUnit)

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* [Cloning of the Parts](#CloningHMParts)

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* [Fluorescence Visualization](#FluorescenceVisualizationHM)

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* [Flow Cytometry](#FlowCytometryHM)

  ---
* [Summary](#ResultsSummary)

  ---
* [References](#results-refs)

{% endblock %}