iGEM 2024 — Team kuleuven

{% extends "layout.html" %}
{% block css %}
{%endblock%}
{% block title %}Notebooks{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}
[Wetlab Notebook](#Wetlab)
 [Drylab Notebook](#Drylab)

# Wetlab Notebook

When working in the laboratory it is vital to document all the experiments, specially when working in a group, since you might have to continue someone else task. At the beginning of our laboratory work, we recorded each task, the team member who completed it, the date, results, and observations. Later on, we decided to work with mini-projects\* to have a more decentralized and structured way of working. For better tracking, we began organizing information in the notebook according to each mini-project number. On this page, you can find the notebook as well as a description of each mini-project. However, if you want more information about the experiments, check out our  [experiments page](experiments)  .

\*To have more information about our mini-project methodology visit our contributions page  [contributions page](contribution)  .

##### MP#1: Saving Private Ryan

* **Goal:** Most of the short peptide sequences that were introduced by inverse PCR were not positive after transformation. This project aimed to fix those in less than 10 days (about 1 and a half weeks) and detect colonies expressing these short peptides.
* **Dates:** 15/07/2024 -> 24/07/2024
* **Results:** The primers used had mutations so we developed a new strategy based on two overlapping primers with repeated sequences in both ends so that they circularize.

##### MP#2: Pick and Pray

* **Goal:** a correlation between number of colonies and transformation success was seen in our project. Those constructs with zero or little colonies (less than 30) were assigned to the MP1, while those that had a high number of colonies were screened again by selecting more colonies (short protein 10).
* **Dates:** 15/07/2024
* **Results:** we did not get any results. The project ended up stopping since we had mutations in the short peptides and the effort to fix them was not worth it.

##### MP#3: Spearhead

* **Goal:** The cloning of the linker was of great importance because it allowed for the cloning of gblocks that we had received by that time. The goal was to prioritize the process of cloning for the linker, following the same steps as in the MP1.
* **Dates:** 15/07/2024 -> 16/07/2024
* **Results:** The linker was cloned successfully but after Sanger sequencing analysis we discovered some mutations in the sequence that lead to a frame shift and ultimately made it useless.

##### MP#3.5: Spearhead.5

* **Goal:** The goal was to rapidly clone the gblock modA (that binds Mo), as the drylab team could use the data generated in the quantification to perform models and enhance the activity of modA.
* **Dates:** 16/07/2024 -> 30/07/2024
* **Results:** Positive results showing the correct insertion of the gBlock were seen after Sanger Sequencing. However, the linker was still mutated even after using a new primer set for amplification, so the sequences needed to be fixed by (The first time all the obtained colonies were negative).

##### MP#3.9: Spearhead.9

* **Goal:** ModA turned out to be not as easy to modulate because of the lack of tools to effectively predict Mo ions binding. Instead, HRMBO, Landmoduling and SilE were prioritized, as Ni and lanthanides seemed to fit our expertise better.
* **Dates:** 29/07/2024 -> 02/08/2024
* **Results:** HRMBP, Landmodulin, and SilE were introduced into the gBlock protein library. A miniprep for gblocks 13.4, 14.4, 15.4 (HRMBP, Lanmodulin, SilE) and sending for Sanger sequencing, where we achieved a positive construct of SilE and HRMBP. Landmodulin was discarded after this.

##### MP#4: Show must go on

* **Goal:** The goal of this miniproject was to continue with the construct that was detected via PCR and prepare O/N cultures for performing a miniprep and sending the plasmids to sequence.
* **Dates:** 15/07/2024 -> 22/07/2024
* **Results:** Sanger sequencing showed some mutations for the successful inversed PCR of the short peptides. A new strategy for the cloning of these needed to be performed. A few of these proteins did not have any mutations. These were used for the preparation of O/N culture after checking in arabinose plates if there was any GFP presence. The O/N cultures were used for the preparation of glycerol stock on the next day.

##### MP#5: Is MBP (p)BAD for you?

* **Goal:** The goal was to substitute sfGFP for MBP upstream, the cloning site of the short peptides and gblocks. To do so an overhang PCR was performed for the MBP sequence followed by Gibson Assembly of the MBP into the sdGFP p BAD vector.
* **Dates:** 15/07/2024 -> 19/07/2024
* **Results:** MBP was correctly inserted into the pBAD vector and substituted GFP, as we could see by colony PCR, sanger sequencing and arabinose induction (no GFP induction was seen).

##### MP#6: Behead Bsal

* **Goal:** Once MBP was cloned into pBAD, a BsaI cut site was detected inside the MBP sequence. To eliminate it, a SDM (Side Directed Mutagenesis) PCR was performed. After transformation, an endonuclease assay was performed using the original MBP pBAD sequence and comparing it to the BsaI mutated cut site.
* **Dates:** 19/07/2024 -> 30/07/2024
* **Results:** Three of the four samples successfully removed the Bsal cut site, as we showed in the next assay.

![BsaI restriction](https://static.igem.wiki/teams/5060/bsai.png)

Figure 1: Restriction with BsaI, project MP#6. The last three sample do not present a BsaI so the SDM worked

##### MP#7 and MP#10: A new horizon

* **Goal:** Once MBP was free of internal BsaI cut sites, it was ready to be implemented into our system for protein expression. The positive results obtained through the Sanger sequencing on MP#4 could be amplified using overhang PCR and introduced by Gibson Assembly into the MBP pBAD vector.
* **Dates:** 25/07/2024 -> 22/08/2024
* **Results:** Although the MP was started early and the first PCR to introduce overhangs for the Gibson Assembly was already done, we decided that there was no point in doing this project, as our solution was now centered on in-vivo remediation (using the cells) instead of using purified proteins, which was the main point of using MBP as the carrier for our protein.

##### MP#8: Catch me if you can

* **Goal:** The gBlocks containing our SpyTag system finally arrived, and we started the process of cloning into pBAD and pBAD2. PBAD2 was supposed to be a plasmid with a compatible ORI and a different selection marker (cm) than pBAD (kan). The idea was to co-transform a culture so it could produce both monomers required for the polymerization of the system.
* **Dates:** 17/07/2024 -> 05/09/2024
* **Results:** Although we had some difficulties with this project, we were able to clone into pBAD. Unfortunately, we had to drop pBAD2 since it was not working.

##### MP#9: I can fix him

* **Goal:** The sanger sequencing from MP#1 and MP#3 indicated the presence of variable mutations on the short peptides (those introduced by inverse PCR). The goal of the project was to use new specific primers designed for SDM for each of the proteins to “fix them”.
* **Dates:** 29/07/2024 -> 14/08/2024
* **Results:** Several strategies were used to perform this SDM, the final result was overall positive, as most of the proteins were successfully cloned into the pBAD plasmid.

##### MP#9.5: I can fix him.5

* **Goal:** After the cloning of the linker downstream sfGFP in pBAD, a deletion was detected upon receiving Sanger sequencing results. This deletion translated into a frameshift that made it impossible for the expression of the gBlocks proteins cloned downstream of the linker. The goal here was to use a SDM primer set to fix each of the gBlocks.
* **Dates:** 1/08/2024 -> 16/08/2024
* **Results:** Several strategies were used to perform this SDM, the final result was overall positive, as most of the proteins were successfully cloned into the pBAD plasmid.

##### MP#11: Please work

* **Goal:** Development and standardization of a toxicity assay for different types of metals and concentrations. The idea was to use it as a screening platform that could be adapted to our protein library.
* **Dates:** 22/07/2024 -> 05/09/2024
* **Results:** The toxicity assay left a bitter taste, as a lot of days and effort were put into this. However, it was only at the end of the project that the results started to make sense and be reproducible. Overall, this MP showed the toxic ranges for *E. coli* of the metals we were working with.

##### MP#12: Golden gate for Short peptides

* **Goal:** New strategy for cloning short peptides that could not be cloned or fixed beforehand. It was based on available online information and PI counseling. The idea was to include overhangs that codify for BsaI cut sites in both flanks of the inverse PCR part, with the intention of digesting them after PCR and using the overhangs as anchoring points for a more efficient ligation, hoping that the mutations in the middle of the sequence would stop.
* **Dates:** 25/07/2024 -> 19/08/2024
* **Results:** The results of this new strategy were overall good, as all short peptides that could not be cloned during MP1 or MP4 were now cloned and expressed successfully.

##### MP#13: Piggy bank

* **Goal:** Small MP with the aim of preparing and labeling glycerol stocks for the high number of positive constructs we were achieving by that point. Glycerol stocks allowed an easy way of preparing standardized O/N cultures.
* **Dates:** 06/08/2024 -> 08/08/2024
* **Results:** The glycerol stocks were successfully prepared and labeled.

##### MP#14: Safety form

* **Goal:** Small MP with the aim of completing the iGEM deliverable for the safety form.
* **Dates:** 07/08/2024
* **Results:** The safety form was completed and submitted on time.

##### MP#15: Positive in Tag

* **Goal:** The aim was to clone the positive results (both gBlocks and short peptides) into the tag sequence (by substituting sfGFP for the coding sequence of the metal binding protein). This would be done by classic restriction enzyme cloning.
* **Dates:** 12/08/2024 -> 26/08/2024
* **Results:** This project was put on pause due to the cloning of the catcher taking longer than expected. Only a few constructs were inserted into the tag sequence.

##### MP#16: Glycerol stock

* **Goal:** Small MP with the aim of preparing and labeling glycerol stocks. Glyerol stock (P1, J140,MymT,PdBP, GolB, MT\_SYNSP, MT, ArsR, Azurin, Csp1, HRMBP, SilE, ModA).
* **Dates:** 20/08/2024
* **Results:** The glycerol stocks were successfully prepared and labeled.

##### MP#17: Third chance for the gBloks

* **Goal:** Cloning some gBlocks that could not be fixed by SDM in MP 9.5. To do so, the correct linker sequence (fixed) was used this time. The cloning protocol was a classic Golden Gate and was used for Csp1 and for the gBlocks received by the dry lab, with mutated sequences that could enhance the activity of the protein. These were Csp1.3, Csp1.8 and SmtB.7.
* **Dates:** 19/08/2024 -> 04/09/2024
* **Results:** The gBlocks were easily cloned using the golden gate strategy by using the linker sequence from an already corrected protein. The drylab sequences were correctly cloned after a second attempt (the first time all of the cloned sequences corresponded to the ModA sequence, indicating a possible mixture of labels or tubes).

##### MP#18: Cool plates

* **Goal:** A new screening platform needed to be implemented to test for the metal binding capabilities of our protein library because the toxicity assay was not completely matured by this point. The goal was to develop and standardize a protocol for creating agar plates with a gradient of metal concentration.
* **Dates:** 20/08/2024 -> 09/09/2024
* **Results:** We developed a new method that can cherry-pick the fittest bacterial strain against a certain metal concentration. The agar plates preparation was standardized, and we switched to spot-assay to improve results visualization.

##### MP#19: Colorimetric assay

* **Goal:** In our pursuit of measurement mention, we aimed to develop a high throughput method for Copper detection based on colorimetric approaches. This method was meant to quantify the Copper recuperation from a lab water sample containing high levels of Cu on it, and use it as benchmark for the different proteins of the library.
* **Dates:** 20/08/2024 -> 09/09/2024
* **Results:** We adapted a previously reported assay from the literature as an alternative to purchasing a commercial kit, successfully establishing linearity in our standard curve for the concentration range of interest. Although we obtained valuable results, there were challenges during the assay preparation that need further optimization to improve its robustness.

During our wet lab work, we used Benchling’s digital logbook as our primary tool for documentation. In addition to this digital notebook, we maintained a physical one, where we mainly added sequencing labels to keep track of all the proteins we sent for sequencing. To complement these, we also used a shared folder on Drive to organize and store photos of the PCR gels, toxicity assays, colorimetry results and other data that is too complex to include in the digital notebook.

You can explore the referenced digital notebook below:

Dowload the pdf  [here](https://static.igem.wiki/teams/5060/logbook-benchling-1.pdf%20)  if it does not show in your browser

# Drylab Notebook

Like in wet lab our work was organized between mini-projects, with each project being distributed throughout the dry lab team. These projects were organized in our dry lab notebook where our results and other information were noted down. On this page, you can find the notebook and description of each mini-project.

##### MP#1: Protein Spearhead Choice

* **Goal:** Decide on which protein will be used for further experiments.
* **Dates:** 08/07/2024 -> 14/07/2024
* **Results:** Based on evolutionary analysis, structure prediction, and metal availability, Csp1 and SmtB were chosen as the target proteins.

##### MP#2: Binding Site Prediction

* **Goal:** Establish baseline data for metal binding in Csp1 and SmtB.
* **Dates:** 15/07/2024 -> 23/07/2024
* **Results:** Identified the key residues involved in metal binding and simulated docking within the 3-dimensional protein structure.

##### MP#3: Machine Learning Model

* **Goal:** Identify metal-binding features through machine learning.
* **Dates:** 06/08/2024 -> 10/08/2024
* **Results:** The neural network was constructed but the lack of conclusive data from toxicity assays affected the development of an adequate training data set to pursue further results.

##### MP#4: Molecular Dynamics

* **Goal:** Understand the physics behind metal binding in our proteins.
* **Dates:** 06/08/2024 -> 16/09/2024
* **Results:** Modeling metal in forcefields was more complex than anticipated as they form non-covalent bonds, so our simulations did not provide concrete results.

##### MP#5: Binding Optimization

* **Goal:** Optimize metal binding from the wild-type sequences for Csp1 and SmtB.
* **Dates:** 24/07/2024 -> 16/09/2024
* **Results:** Through metal binding analysis software, our mutated sequences (specifically Csp1 #3, 7, and SmtB #8) were seen to show improved binding to their respective metals. These sequences were then sent to wet lab to use as potential enzymes with improved metal-binding capacity.

During our dry lab work, we used Benchling’s digital logbook as our primary tool for documentation. In addition to this digital notebook, we used online browser interfaces of published, open-source metal-binding software (either hosted by the publisher’s domain or ported to Hugging Face, a collaborative machine-learning community).

You can explore the referenced digital notebook below:

Dowload the pdf  [here](https://static.igem.wiki/teams/5060/drylabnotebook.pdf%20)  if it does not show in your browser

{%endblock%}

{% extends "layout.html" %}
{% block css %}
{% endblock %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}
[Introduction](#Intro)
[Cycle 1](#Cycle1)
 [Cycle 2](#Cycle2)
 [Cycle 3](#Cycle3)
 [Cycle 4](#Cycle4)

## Engineering Cycles

**Cycle 1**

##### Characterization of metal binding proteins

**Cycle 2**

##### Resolving mutated deletions

**Cycle 3**

##### Gradient toxicity assays

**Cycle 4**

##### Spytag - Spycatcher

## Engineering cycles summary

![Example of an engineering cycle used in this page](https://static.igem.wiki/teams/5060/desing-build-test-learn-engineering.png)

Figure 1: The design-build-test-learn cycle.

Synthetic biology, often referred to as bioengineering, applies engineering principles to the development of biological systems, ensuring they perform the desired function. This approach mirrors the method engineers use to develop products—through an iterative process of design, testing, and refinement. In synthetic biology, this method is known as the **Engineering Cycle**, and it is repeated until the biological system behaves as intended.

The Engineering Cycle consists of four distinct phases: (1) **Design**, (2) **Build**, (3) **Test**, and (4) **Learn** (Fig.1). Each phase brings us closer to achieving a biological system capable of solving real-world challenges.

Throughout the execution of our project, we adhered to a structured workflow based on the Engineering Cycle to guide each phase of development (Fig.1). Whenever we faced an **obstacle**, we analyzed it thoroughly, redesigned components where necessary, and iterated on our workflow to build solutions that met the evolving requirements. By progressing through the design, build, test, and learn phases, we were able to refine our project in a **logical** and **methodical manner**. Testing yielded valuable insights, which we integrated back into the design, constantly optimizing our biological system to overcome any hurdles. In this way, the Engineering Cycle was not just a theoretical framework but a dynamic process that empowered us to adapt, troubleshoot, and improve as we progressed.

Throughout our project, we identified several key engineering cycles that were critical in achieving our milestones (Fig.2):

1. Choice of **metal binding proteins** and improving their binding properties.
2. **Cloning** and **expression** of metal-binding constructs – Inserting the peptides into bacterial strains.
3. Development of **colorimetric and toxicity assays** – Optimizing assays to test the metal accumulation efficiency.
4. **Polymerization** of Spytag and Spycatcher

![Fig 2: Overview of the 4 engineering cycles and how they interact.](https://static.igem.wiki/teams/5060/4-engineeringcycles.png)

Figure 2: Overview of the 4 engineering cycles and how they interact.

The identified key engineering cycles are closely interconnected, The first interaction occurs between Cycle 1 and Cycle 2. In the build phase of Cycle 1, mutations were encountered during cloning, which Cycle 2 focuses on resolving. The final learnings of cycle 2 was brought back to complete the cloning in the build phase of Cycle 1.

The second interaction is between Cycle 1 and Cycle 3. When testing the cloned bacteria for metal-binding activity in Cycle 1, improvements were needed in the gradient assay, this was the baseline of Cycle 3. The testing phase in Cycle 1 influenced the design of Cycle 3’s experiments. The final insights from Cycle 3 are then applied back to refine the testing phase of Cycle 1.

There is no direct interaction between Cycle 4 and the previous cycles, as it followed an independent pathway. The diagram (Fig.2) visually represents these interactions, showing how the cycles interrelate to refine the project’s outcomes at each phase.

## Cycle 1 - Characterization of Metal Binding Proteins

In this cycle, we focus on bridging **computational modeling** (Dry lab) and **experimental validation** (Wet lab) within our project. By creating a feedback loop, we allow computational predictions to inform our experiments, while the outcomes from these experiments refine our future computational analyses. This integrative approach enables us to iteratively optimize our systems and enhance the performance of our metal-binding peptides.

### Cycle 1.1

## Click a section to learn more! In this cycle, the Test and Learn sections are the same

Test
Learn
Design
Build

### Cycle 1.2

## Click a section to learn more! In this cycle, the Redesign and Build sections are the same!

Test
Learn
Redesign
Build

## Cycle 2 - Resolving Mutated Deletions

During the construction of our protein expression systems, we aimed to clone several genes of interest into a pBAD plasmid. However, during this process, we encountered **unexpected mutations** resulting from blunt-end ligation. These mutations affected two distinct regions: for short peptides expression vectors, mutations were found within the short peptide gene itself, while for larger proteins expression vectors, the mutations occurred in the linker region between the target protein and the sfGFP tag .

These errors posed significant challenges, as they caused **frameshift mutations**, leading to out-of-frame translation. This cycle focuses on resolving these point mutations and restoring correct in-frame translation for functional protein expression.

![genetic construct for screening the metal binding proteins, annotated with a point deletion.](https://static.igem.wiki/teams/5060/engineering-figuur-3.png)

Figure 3: genetic construct for screening the metal binding proteins, annotated with a point deletion.

![genetic construct for screening the metal binding peptides, annotated with DNA deletion.](https://static.igem.wiki/teams/5060/engineering-figuur-4.png)

Figure 4: genetic construct for screening the metal binding peptides, annotated with DNA deletion.

### Cycle 2.1

## Click a section to learn more!

![Phase image]()

Test
Learn
Design
Build

### Cycle 2.2

## Click a section to learn more!In this cycle the Redesign and Build sections are the same!

![Phase image]()

Test
Learn
Redesign
Build

## Cycle 3 - Gradient toxicity assays

To assess whether metal-binding proteins can enhance the survival rate of *E. coli* under metal stress, we designed an experiment using a **gradient agar plate** with increasing concentrations of metal ions. The rationale behind this approach was to expose *E. coli* to progressively harsher metal conditions, thereby allowing us to observe any potential protective effects conferred by the expression of metal-binding proteins. By comparing the growth and survival of *E. coli* strains with and without these proteins, we aimed to determine their **effectiveness in metal tolerance**, providing valuable insights into their functional role in metal binding and detoxification.

### Cycle 3.1

## Click a section to learn more!In this cycle the Design and Build sections will be the same!

![Phase image]()

Test
Learn
Design
Build

### Cycle 3.2

## Click a section to learn more!In this cycle the Redesign and Build sections will be the same!

![Phase image]()

Test
Learn
Redesign
Build

## Cycle 4 - Spytag - Spycatcher

### Cycle 4

## Click a section to learn more!

![Phase image]()

Test
Learn
Design
Build

## References

[1] Trehan, A., Kiełbus, M., Czapinski, J. et al. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.  *Sci Rep 6*, 19121 (2016). <https://doi.org/10.1038/srep19121>

[2] Gahlot, D.K., Taheri, N., Mahato, D.R. et al. Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper.  *Sci Rep 10*, 20327 (2020). <https://doi.org/10.1038/s41598-020-76178-z>

{% endblock %}

{% extends "layout.html" %}
{% block css %}
{% endblock %}
{% block title %}Experiments{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}
[Design & Cloning](#Design&Cloning)
[Metal Binding Assays](#Metal Assays)
[Polymerization](#Polymerization)
[References](#References)

## Design & Cloning

## We cloned a library of 28 proteins!

### Cloning Strategies

The final objective was to  **clone a small library of metal-binding proteins** to evaluate their capacity to bind metals. Two distinct cloning strategies were employed, contingent on the length of the protein. The sequence was introduced via a **PCR devoid of restriction enzymes** for peptides comprising fewer than 50 amino acids (aa). In contrast, for proteins exceeding 50 aa, the restriction enzyme cleavage site was initially introduced via a **PCR**, followed by **a Golden Gate assembly**. Note that all our enzymes came from New England Bioscience. The complete list of proteins is available on the  [Parts page](parts) .

![Image of pBad_sfGFP plasmid ](https://static.igem.wiki/teams/5060/001-pbad-sfgfp.jpg)

Figure 1: pBad\_sfGFP plasmid

### Plasmid Extraction

**DH10B**  cells harboring the  **pBAD\_sfGFP**  were grown overnight with kanamycin (50 µg/mL) in a Luria-Bertani (LB) medium. It contains an inducible araBAD promoter followed by sfGFP carrying Kan resistance gene. The tubes were centrifuged at 5000 x g for 15 minutes at 4°C. The pellet was recovered. Following the manufacturer's procedure, the plasmid was extracted from the cells using a  **Thermofisher Scientific GeneJET Plasmid Miniprep Kit** . The dsDNA concentration and the purity rations (260/230 and 260/280) were measured using the  **SPECTROstar Nano**  (BMG Labtech). Prof. Vitor Pinheiro donated the empty backbone plasmid.

### Primers Design

The primers were designed using  **Benchling**  and ordered from  **Integrated DNA Technologies (IDT)**. Recognition sites for  **BsaI-HF®v2**  or  **PaqCI®**  were added. Overhangs were incorporated depending on the cloning strategies used. For instance, to perform a Gibson assembly, the overhangs of the primer used to amplify our insert are homologous to the overhang of the primer used to amplify our pBAD. The annealing temperature Tm was calculated using the  **NEB calculator**.

A complete spreadsheet of all primers used can be found in the pdf  [here](https://static.igem.wiki/teams/5060/003-list-of-primers.pdf)

### PCRs

We amplified our short peptides, gBlocks™, linker and plasmids using  **Taq DNA Polymerase (NEB)**  and  **Q5 High Fidelity DNA Polymerase (NEB)**. These PCR reactions were conducted according to the manufacturer’s guidelines:

##### Table. 1 PCR Components and Volumes for 25 µl and 50 µl Reactions Using Taq DNA Polymerase with Standard Buffer

|  |  |  |  |
| --- | --- | --- | --- |
| Components | 25 uL Reaction | 50 uL Reaction | Final Concentration |
| 10X Standard Taq Reaction Buffer | 2.5 uL | 5 uL | 1X |
| 10 mM dNTPs | 0.5 uL | 1 uL | 200 uM |
| 10 uM Forward Primer | 0.5 uL | 1 uL | 0.2 uM (0.05-1 uM) |
| 10 uM Reverse Primer | 0.5 uL | 1 uL | 0.2 uM (0.05-1 uM) |
| Template DNA | Variable | Variable | <1,000 ng |
| Taq DNA Polymerase | 0.125 uL | 0.25 uL | 1.25 units/ 50 uL PCR |
| Nuclease-Free Water | Up to 25 uL | Up to 50 uL |  |

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time |
| Initial Denaturation | 95°C | 30 seconds |
| 30 cycles | 95°C | 15- 30 seconds |
| 45-68°C | 15- 60 seconds |
| 68°C | 1 minute/kb |
| Final Extension | 68°C | 5 minutes |
| Hold | 4-10°C |  |

PCR Components and Volumes for 25 µl and 50 µl Reactions Using Q5 High-Fidelity DNA Polymerase

|  |  |  |  |
| --- | --- | --- | --- |
| Components | 25 uL Reaction | 50 uL Reaction | Final Concentration |
| 5X Q5 Reaction Buffer | 5 uL | 10 uL | 1X |
| 10 mM dNTP | 0.5 uL | 1 uL | 200 uM |
| 10 uM Forward Primer | 1.25 uL | 2.5 uL | 0.5 uM |
| 10 uM Reverse Primer | 1.25 uL | 2.5 uL | 0.5 uM |
| Template DNA | Variable | Variable | <1,000 ng |
| Q5 High-fidelity DNA Polymerase | 0.25 uL | 0.5 uL | 0.02 U/uL |
| Nuclease-Free Water | Up to 25 uL | Up to 50 uL |  |

|  |  |  |
| --- | --- | --- |
| Step | Temperature | Time |
| Initial Denaturation | 98°C | 30 seconds |
| 30 cycles | 98°C | 5- 10 seconds |
| 50-72°C | 10- 30 seconds |
| 72°C | 20- 30 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4-10°C |  |

### Gel Electrophoresis

Gel electrophoresis was carried out to separate and visualize our amplified DNA based on its size. It is also a way to assess the success of our PCR. Depending on the length of our DNA fragments, we prepared agarose gel with the optimal resolution according to the manufacturer’s labels. The procedure is described as follows:

1. Mixing the adequate amount of agarose powder with 50ml of  **1X lithium acetate.**
2. The mixture must be microwaved until complete dissolution.
3. Add 3 μL **SBYR safe** and pour everything into a gel tray with the well comb in place. Make sure there are no bubbles and wait until gel solidification at room temperature.

The next step is sample loading which is performed as follows:

1. 5 uL of DNA was added to 1 uL of **6X Loading buffer**. The sample was then collected and loaded in the wells.
2. For our shortest peptides, we used a Low Molecular Weight DNA Ladder. Whereas, for our gBlocks™ and plasmids, we used either a  **1 kb DNA Ladder** or a  **1 kb plus DNA Ladder**. All ladders were acquired from NEB.

### DpnI digestion

Once our PCR products were ready, the methylated strands were digested with **DpnI** for 1 hour at 37°C to select for the newly synthesized DNA strands.

### DNA Purification

We extracted our PCR products from the reaction using a **GeneJET PCR purification kit** from Thermo Fisher Scientific according to the manufacturer's instructions. The principle behind this procedure is that the mixture containing DNA is mixed with the binding buffer and passed through the GeneJET Purification Column. The binding buffer contains a chaotropic agent that denatures proteins and promotes the binding of the DNA to the silica membrane inside the column. Any impurities are removed in the washing step. The purified DNA is eluted from the column using the elution buffer. Finally, the quantity and the quality of the purified DNA is assessed using the **SPECTROstar Nano LVis Plate** (BMG LABTECH).

### Phosphorylation

44 uL of the digested purified DNA is mixed with 1 uL of **T4 PNK** and 5 uL of **10X Buffer DNA ligase** (10 mM ATP). The mixture is then incubated at 37°C for 1hr and heat inactivated by incubating at 80°C for 20 minutes.

### Ligation

In our 55 uL sample, 1 uL of the  **T4 DNA ligase**  is added. The sample is incubated for 2 hours at room temperature or kept in the thermocycler overnight at 16°C.

### Transformation

After obtaining our correct genetic material, chemically competent **DH10B** cells were used (see below). 50 uL of competent cells that were previously stored at - 80°C were thawed on ice and 5 uL of the cloning reaction mix were added. The tubes were kept on ice for 30 min. Then, the cells were **heat shocked** at 42°C for 30 seconds and were **immediately chilled** on ice for 5 min. Then, 450 uL of prewarmed sterile **LB** medium was added. The tubes were incubated with shaking at 37°C for 1 hour. Lastly, 50 uL of the sample was plated on LB supplemented with kanamycin.

### Colony PCR

After transformation, plates were checked the next day, and single colonies were picked using a disposable inoculation loop and washed in a PCR tube containing 10 uL of sterile water. Afterward, using the same loop, it was streaked on a **“replica” plate** containing LB and kanamycin. The plates were incubated at 37°C overnight. The cells in the PCR tubes were **lysed** at 95°C for 2 minutes using a thermocycler. Next, the debris was centrifuged at 2000 x g for 5 minutes. The supernatant was recovered and used as a template to perform a **Taq polymerase PCR** under the appropriate conditions. The right primers were used for the amplification and the length of the amplicon was checked using agarose gel. The samples were then sequenced following Macrogen Ez-Seq instructions.

### Glycerol stock

Glycerol stock was prepared to store bacterial cultures for the long term. First, overnight liquid cultures were prepared using **LB and adequate antibiotic resistance** and incubated at 37°C overnight with shaking. After having bacterial growth, 900 uL of the liquid culture was added to 900 uL of **40% glycerol stock** in a cryovials and mixed gently. The tubes were stored in –80 °C.

### Golden Gate

We used Golden Gate assembly to **simultaneously and directionally assemble** the fragments of our vector, linker and blocks. We accounted for a **2:1 ratio of insert: backbone**. The procedure was carried out as follows

|  |  |
| --- | --- |
| Components | 10 uL reaction |
| Backbone | Variable |
| Insert | Variable |
| T4 DNA Ligase Buffer 10X | 1 uL |
| T4 DNA Ligase | 0.5 uL |
| BsaI-HF®v2 | 0.5 uL |
| Nuclease-Free Water | Up to 10 uL |

### Competent Cell Preparation:

The cells used in the transformation were chemically competent cells. First, 5 mL of overnight cultures of DH10B cells were prepared. Next, **2 mL of the bacterial culture (1/100 volume) was diluted into 200 mL of freshly prepared LB**. It was incubated at 37°C until it reached **OD600 = 0.4**. The total culture was split into 4 falcon tubes (50 mL) and pelleted at 4000 x g for 15 minutes at 4°C. After discarding the supernatant, the cells were resuspended in total 50 mL cold **MgCl2** (12.5 mL of MgCl2 in each falcon tube). **The resuspension was done by gently tapping the falcon tubes on the hand palm without pipetting up and down**. The cells were centrifuged at 4000 x g for 15 minutes at 4°C. The cells were gently resuspended again using **CaCl2** in a total volume of 50 mL as previously done. Then, the tubes are placed on ice for 30 min and centrifuged again. Afterwards, the pellets resuspended in 2.5 mL of **85 mM CaCl2, 15% glycerol**. The content of the falcon tubes is combined and centrifuged. The cells are resuspended gently in 2 mL **85 mM CaCl2, 15 % glycerol**. Finally, a 50 uL aliquot was added into sterile 1.5 mL Eppendorf tubes while everything is kept on dry ice. The competent cells are stored in –80 °C.

## Metal Binding Assays

## We screened our proteins with 3 different assays!

### Spot Assay

Overnight cultures of bacterial strains containing our expressed metal-binding proteins and control are prepared using 5 mL of LB with kanamycin. A gradient agar plate is prepared as follows [1]. **In a square petri dish (12cm x 12cm), two batches of LB are poured**. The first LB Batch (50 mL) is **LB mixed with the highest metal concentration** recommended by literature (usually 1000 mg/ L) and the second batch is LB with **kanamycin and 0.02% of L-arabinose**. The bottom of the square petri dish is marked at 0.9 cm from the bottom of the plate to indicate where the LB batch should reach. The metal-containing LB agar is poured first, tilting the dish to the mark, and allowing it to solidify before adding the LB layer.

![Fig.2  Gradient agar preparation. We thank IDT for their “support”! ](https://static.igem.wiki/teams/5060/agar.png)

Figure 2: Gradient agar preparation. We thank IDT for their support!

After overnight incubation of the cultures, the optical density OD600 is measured and diluted to 0.2, and 0.02% arabinose is added to induce the expression. The cells are incubated again for 2- 3 hours. The OD is measured again, and the cultures are diluted to **10^6 cells/ mL** at certain concentrations, and **3uL are plated onto the gradient agar plates**. The latter are incubated overnight. The next day, the plates were scanned using the Typhoon™ FLA 9500 biomolecular imager at the correct wavelength to see the fluorescence of sfGFP expressed by the cells.

![Fig. 3 Gradient agar plate with the highest concentration of Cu2+ on the left and no metal on the right ](https://static.igem.wiki/teams/5060/20240912-cu-agar-plate-flipped-plate-1.png)

Figure 3: Gradient agar plate with the highest concentration of Cu2+ on the left
and no metal on the right. To see more results, check our Results page!

### Toxicity Assay

Overnight cultures of bacterial strains containing our expressed metal-binding proteins and control are prepared using 5 mL of LB with kanamycin. The next day, the optical density is measured and diluted to 0.2. 0.02% of arabinose is added to induce the expression and the cultures are incubated for 2- 3 hours. Meanwhile, dilutions of the metal solutions to be tested are prepared: **10, 50, 75, 100, 250, 500, 750, and 1000 mg/L**. After incubation is done, the optical density is checked again and converted to find the concentrations of cells. The latter is diluted until obtaining 1000 cells/ mL. A 96-well microplate is used where each well contains a total volume of 200 uL: **100 uL is the metal solution + 100 uL is the bacterial culture**. Using the spectrophotometer, a reading is performed to collect the initial data. The microplate is incubated with shaking at 37°C with shaking. A second reading is performed after incubation.

![Fig. 4 Schematic representation of a microplate used to perform high-throughput toxicity assays](https://static.igem.wiki/teams/5060/schematic-microplate.png)

Figure 4: Schematic representation of a microplate used to perform high-throughput toxicity assays. To see more results, check our  [Results page](results) !

### Copper Colorimetric Assay

Similar to the toxicity assay, overnight cultures of bacterial strains containing our expressed metal-binding proteins and control are prepared using 5 mL of LB with kanamycin. The next day, the optical density is measured and diluted to 0.2. 0.02% of arabinose is added to induce the expression and the cultures are incubated for 3 hours. Meanwhile, dilutions of the metal solutions to be tested are prepared. In our colorimetric assay, we used three main reagents: **ubiquinone, hydroxylamine hydrochloride, and acetate buffer**. The biquinodone was prepared by mixing 0.02 g of biquinodone, and 100 uL of 100% triton-X in 50 mL of 70% ethanol. The sample is placed in a sonification match for 2 minutes and stirred with a magnetic stirrer at 40°C for 5 minutes. The Hydroxylamine Hydrochloride Solution was prepared by dissolving 0.75M of hydroxylamine hydrochloride in 20 mL of demineralized water. The acetate buffer is prepared by mixing 0.1 M of sodium acetate and 0.089 mL of acetic acid 99% in 50 mL of distilled water. pH is adjusted to 4.8 using 10N HCl.

After incubation, our cells are centrifuged at 9000 xg for 5 minutes. After discarding the supernatant, the cells are washed using 0.85% NaCl and centrifuged again. The cells are resuspended in **0.85% NaCl** and the OD is measured to get the concentrations of cells. Using the 0.85% NaCl solution, cell culture, and metal stock solution, a dilution to reach **10^8 cells/mL** and the correct concentration of the metal to be tested is performed. The samples are incubated for 1 hour and centrifuged again. The supernatant is collected. The samples are prepared as follows: 200 uL of supernatant, 200 uL of hydroxylamine hydrochloride, 1 mL of 2 2'-biquinoline, and 200 uL of acetate buffer. A blank is prepared too for comparison purposes: 200 uL of 0.85% NaCl, 200 uL of hydroxylamine hydrochloride, 1 mL of 2 2'-biquinoline, and 200 uL of acetate buffer. The tubes are centrifuged for 3 min at 11.5 xg. Finally, **250 uL of the supernatant is collected and plated on the 96-well microplate**. The absorbance of the resulting solutions was measured using a spectrometer at 545 nm.

![Fig. 5 Cuvettes to perform a colorimetric assay.](https://static.igem.wiki/teams/5060/pink-cuvets.jpg)

Figure 5: Cuvettes to perform a colorimetric assay. To see more results, check our  [Results page](results) !

### Cobalt Standard Curve Preparation

The standard curve preparation started by dissolving cobalt II chloride to prepare a main stock solution of 4000 mg/L. From the latter, we prepared 1.5 mL of eight standard solutions with the following concentrations: 10 mg/L, 25 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, and 300 mg/L. For each standard, 200 uL of the solution is added to 80 uL of 6M hydrochloric acid, 200 ML of 50% potassium thiocyanate (KSCN), and 480 uL of acetone [2]. After thorough and careful mixing, the absorbance of each sample is measured using a spectrophotometer at 620 nm.

The absorbance values are recorded and plotted against the corresponding cobalt concentrations to create a
standard curve. The latter is used as a reference for determining unknown cobalt concentrations by correlating their absorbance with the linear relationship established in the plot.

## Polymerization

## We made a linear polymer with SpyTag and SpyCatcher

In addition to the metal-binding proteins, we designed and cloned two fluorescent proteins, sfGFP and mCherry, flanked by two **SpyTags** and two **SpyCatchers**, respectively. The goal was to create a polymer capable of absorbing metals and removing them from contaminated water. To verify that polymerization was possible, we expressed
Tag-sfGFP-Tag and Catcher-mCherry-Catcher individually, lysed the cells and combined the two lysates to make polymerization take place. Afterwards, we ran an SDS-PAGE gel to check the molecular weight of the products. Finally, we scanned the gel at a wavelength of 473nm to excite both fluorescent proteins

![Fig. 6 SDS-PAGE of Tag-sfGFP-Tag and Catcher-mCherry-Catcher](https://static.igem.wiki/teams/5060/sds-page-t-sfgrp-t-and-c-mcherry-c.png)

Figure 6: SDS-PAGE of Tag-sfGFP-Tag and Catcher-mCherry-Catcher. Samples were prepared using two different temperatures. Lane 2 was scanned with different exposure to see the pattern at higher molecular weight.

Lane 1 ladder

Lane 2 TsfGFPT + C-RFP-C (37°C)

Lane 3 TsfGFPT (95°C)

Lane 4 C-RFP-C (95°C)

Lane 5 TsfGFPT + C-RFP-C (95°C)

Lane 6 pellet TsfGFPT (95°C)

Lane 7 pellet C-RFP-C (95°C)

Lane 8 TsfGFPT (37°C)

Lane 9 C-RFP-C (37°C)

### References

[1] Gahlot, D.K., Taheri, N., Mahato, D.R. et al. Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper.  *Sci Rep 10*, 20327 (2020).  [https://doi.org/10.1038/s41598-020-76178-z](https://doi.org/10.1038/s41598-020-76178-z%20%20)
Rice University. (n.d.). Golden Gate Assembly. Bennett Lab Wiki. https://wiki.rice.edu/confluence/display/BIODESIGN/Golden+Gate+Assembly

[2] NTU (n.d.).  *Quantitative analysis of cobalt (II) ions*   [https://teaching.ch.ntu.edu.tw/gclab/en/pdf/manual/10\_E10-M-Cobalt(II)-ions-2021.02.05.pdf](https://teaching.ch.ntu.edu.tw/gclab/en/pdf/manual/10_E10-M-Cobalt%28II%29-ions-2021.02.05.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block css %}
{% endblock %}
{% block title %}Results{% endblock %}
{% block lead %}In our project, we explored the effectiveness of various metal-binding peptides in enhancing the resistance of *E. coli* against heavy metals like **copper** (Cu²⁺), **nickel** (Ni²⁺), and **zinc** (Zn²⁺) and their metal removal capabilities. By utilizing gradient agar assays and colorimetric analysis, we aimed to assess the survival rates and **metal-removal efficiency** of the engineered peptides. Through this process, we successfully cloned 28 metal-binding peptides and two polymer-forming proteins. Our results indicated that several peptides demonstrated **improved survival rates** and metal-removal capabilities, particularly under copper and nickel stress. For more information on the methodologies of the experiments, visit our  [Experiments page](experiments)  . {% endblock %}
{% block page\_content %}
[Summary](#Summary)
[Cloning](#Cloning)
[Agar Plates](#Agar)
[Toxicity Assays](#Toxicity)
[Colorimetric Assays](#Colorimetric)
 [Polymerization](#Polymerization)

## Summary

![](https://static.igem.wiki/teams/5060/results-wiki-icon-1.svg)

#### Cloning

Cloned 28 metal-binding and 2 polymer-forming peptides.

![](https://static.igem.wiki/teams/5060/results-wiki-icon-2.svg)

#### Agar Plate

From 28 peptides, 22 for copper and 20 for nickel have a better survival rate than pBAD.

![](https://static.igem.wiki/teams/5060/results-wiki-icon-3.svg)

#### Toxicity

Our cells die between 100 and 200 mg/L of copper.

![](https://static.igem.wiki/teams/5060/results-wiki-icon-4.svg)

#### Colorimetric

15 out of 30 peptides have a better percentage removal than pBAD and DH10B.

![](https://static.igem.wiki/teams/5060/results-wiki-icon-5.svg)

#### Polymerization

Made a polymer that can be used as a support to absorb metals.

## Experiment Results

### 1.Cloning

From a total of 34 metal-binding peptides, we successfully cloned **28**, which can be seen in Table 1.

To see an overview of the parts, click   [here](parts)   to go to the Parts section.

###### Type

###### Peptide

###### Metal ions

###### Short Peptides

|  |  |
| --- | --- |
| HP | "Cd2+, Cu2+, Zn2+" |
| P1 | Cu2+ |
| P2 | "Zn2+, Cu2+" |
| P3 | Zn2+ |
| CuPept | Cu2+ |
| PdBP | Pd |
| Ag4 | Ag+ |
| J140 | Cd2+ |
| GaPept | Ga |
| GBP1 | Au |
| AsPept | "As3+, Cd2+, Hg" |
| CDS7 | Cd2+ |

###### Type

###### Peptide

###### Metal ions

###### gBlocks

|  |  |
| --- | --- |
| SmtA | Cd2+ |
| GolB | Au |
| MymT | Cu2+ |
| MT\_SYNSP | "Cd2+, Cu2+, Zn2+" |
| fMT | "Cd2+, As3+" |
| HRMBP | "Ni, Zn" |
| MT | "Cd2+, Zn2+" |
| ArsR | As3+ |
| SmtB | "Cd2+, Zn2+" |
| Csp1 | Cu2+ |
| Azurin H117G | "Pd, Pt" |
| PbrR | Pb |
| silE | Ag+ |
| modA | Mo/W |
| SmtB\_7 | "Cd2+, Zn2+" |
| csp1\_3 | Cu2+ |

###### Type

###### Protein

###### Polymerization

|  |
| --- |
| Tag-sfGFP-Tag |
| Catcher-mCherry-Catcher |

Table. 1: Positive peptides table

For the short peptides, we cloned them as PCR overhangs and the proteins with gBlocks and Golden Gate. During the cloning, we had mutations in some of our peptides. To solve that, we used site-directed mutagenesis. For more information on this technique, click   [here](engineering)   to go to the Engineering Cycles section.

Apart from the metal-binding ones, we cloned two proteins, T-sfGFP-T and C-RFP-C, that can form a polymer. These proteins are fluorescent, but we designed a flexible and easy-to-use system to change protein while maintaining the polymerization ability.

## Agar Plates

### 2.1 Gradient Agar Streaking Assay

In the Gradient Toxicity Assay, we aimed to evaluate the robustness of bacterial resistance to increasing concentrations of metals using gradient agar plates. Unfortunately, this experiment was **not robust enough**, as there was significant variability in growth patterns between the plates without the pBAD control. The inconsistency in growth lengths prevented us from obtaining reliable data, and thus no valuable results could be derived from this assay.

### 2.2 Gradient Agar Spot Assay

The Gradient Agar Spot Assay provided **more consistent** and interpretable results. This assay was conducted for copper (Cu²⁺), nickel (Ni²⁺), and zinc (Zn²⁺). We tested the survival rates of various metal-binding proteins compared to the pBad control under increasing metal concentrations. The results for copper and nickel are summarized in the table below:

![Image of pBad_sfGFP plasmid ](https://static.igem.wiki/teams/5060/results-cu.png)
![Image of pBad_sfGFP plasmid ](https://static.igem.wiki/teams/5060/results-ni.png)
![Image of pBad_sfGFP plasmid ](https://static.igem.wiki/teams/5060/results-zn.png)

Figure 1: Results of the gradient agar plate for the metals copper (top), nickel, and zinc (bottom) respectively

###### Peptide

###### Survival rate compared to pBAD (Cu2+)

###### Survival rate compared to pBAD (Ni2+)

|  |
| --- |
| HP |
| P1 |
| P2 |
| P3 |
| CuPept |
| PdBP |
| Ag4 |
| J140 |
| GaPept |
| GBP1 |
| AsPept |
| CDS7 |
| SmtA |
| GolB |
| MymT |
| MT\_SYNSP |
| fMT |
| HRMBP |
| MT |
| ArsR |
| SmtB |
| Csp1 |
| Azurin H117G |
| PbrR |
| silE |
| modA |
| SmtB\_7 |
| csp1\_3 |

|  |
| --- |
| 0 |
| + |
| + |
| + |
| + |
| + |
| 0 |
| / |
| + |
| + |
| + |
| + |
| + |
| + |
| + |
| 0 |
| 0 |
| + |
| + |
| + |
| + |
| + |
| + |
| ++ |
| + |
| ++ |
| + |
| + |

|  |
| --- |
| + |
| + |
| + |
| + |
| + |
| + |
| / |
| / |
| + |
| + |
| + |
| + |
| + |
| 0 |
| + |
| + |
| + |
| + |
| + |
| 0 |
| + |
| + |
| + |
| + |
| 0 |
| + |
| / |
| + |

Table. 2: Results of the gradient agar plate for the metals copper and nickel, 0 indicates that the mutant had the same number of spots as pBad, + and ++ indicate one and two extra growth spots respectively compared to pBAD, and / indicates that something went wrong with this mutant.

---

For zinc, the experiment did not yield conclusive results. The concentration gradient used was too low, resulting in no distinct cut-off point for bacterial growth. Consequently, all the spots produced colonies, making it impossible to differentiate between the metal-binding proteins and the pBAD control. As a result, no valuable data could be obtained for zinc resistance in this assay.

Overall, several metal-binding proteins, such as **modA** and **PbrR**, demonstrated increased survival rates against Cu²⁺, showing enhanced resistance compared to the pBAD control. Most proteins also showed comparable or improved survival against Ni²⁺, indicating potential candidates for further studies in enhancing metal resistance.

## 3.Toxicity Assay

This experiment is done to test the concentration of the metal that the bacteria with die. Because the initial experiments had too high absorbance values, we changed our protocol and the plate reading parameters. For more information, click   [here](experiments)   to check the experiments section.

After changing the settings, we obtain the graph from Fig. 1. In this graph, the higher the absorbance, the higher the viability of the cells.

![Toxicity asssay graph of the absorbance at 600 nm of each peptide in relation to different concentrations of copper.](https://static.igem.wiki/teams/5060/toxicity.png)

Figure 2: Toxicity assay graph of the absorbance at 600 nm of each peptide in relation to different concentrations of copper

In this way, from Fig. 2, we conclude that:

* A sharp decline in absorbance was observed between 100-200 mg/L, where most strains experienced a significant reduction in population, indicating the threshold concentration at which the bacteria could survive in the presence of metal.
* At the second point, **MT-SYNSP** and **MymT** have higher cell growth than pBAD.
* DH10B has a higher cell growth rate at most copper concentrations.
* pBAD empty has a sudden increase of absorbance at 100 mg/L, which suggested a high variability in our data.

We cannot gather more definite data from this experiment because contaminations can easily happen when doing the microplates, which could explain the sudden increases in some of the readings.

##### **Conclusion:** Most of the cell cultures die between 100 and 200 mg/L

## 4.Colorimetric Assay

### 4.1 Copper

This experiment is done with an alcoholic solution of 2,2’-biquinoline/Triton X-100, which forms a complex Cu(I), hydroxylamine hydrochloride and acetate buffer pH 4.8 to create a purple complex with a maximum absorbance of 545 nm [1].

Firstly, we did the standard curve with cuvettes to find the linear relationship, which can be seen in Fig. 3.

![Standard curve for copper in cuvettes](https://static.igem.wiki/teams/5060/standard-curve-cuvettes.png)

Figure 3: The standard curve of the copper colorimetric assay with 0.85% saline solution and with the concentration of copper of 25, 50, 75, 100, 150, 200, 250 and 300 mg/L

From Fig. 4, we conclude that the proteins stabilize around one hour of incubation.

![Colorimetric experiment at different incubation time](https://static.igem.wiki/teams/5060/incubation-time.png)

Figure 4: Colorimetric experiment at different incubation times with an initial concentration of 75 mg/L of Cu and 0.85% saline solution

To optimize the measurement, we used the microplate instead of the cuvettes. The standard curve can be seen in Fig. 5. For the calculation steps, click   [here](measurement)   to go to the Measurement section.

![The standard curve at 0.85% saline solution with different concentrations of copper](https://static.igem.wiki/teams/5060/standard-curve-microplates.png)

Figure 5: The standard curve at 0.85% saline solution with concentrations of copper at 10, 20, 50, 60, 70 and 80 mg/L in a microplate.

From Fig. 6, we see that:

* From the standard curve, the 50 mg/L point is an outlier. We used the concentration of 50 mg/L for the experiment in Figure 5, which could have caused inaccuracies in the results.
* Peptides P3, Ag4, J140, GaPept, GBP1, CDS7, SmtA, MymT, MT-SYNSP, HRMBP, CSP1, ModA, Azurin, SilE and CSP1.3 have a  **higher removal percentage**  than our controls pBAD empty and DH10B.
* CSP1.3, which the  **dry lab optimized** , seems to exhibit a slightly higher removal percentage than the original CSP1.
* HP, P1, control 2 and FMT have a negative removal, so the metal concentration was higher than the initial one. This could be caused by cells bursting during the wash step, leaking the copper from the inside the cell, or there were interferences from the crystals of 2,2’-biquinoline.

![The percentage of metal removal of 11 proteins](https://static.igem.wiki/teams/5060/metal-removal.png)

Figure 6: The percentage of metal removal of 11 proteins, including pBAD and DH10B as controls, with incubation time at 1 hour and initial concentration of 50 mg/L of Copper. Controls 1 and 2 are the saline solution with 50 mg/L of copper to check any criticality during the experiment. Control 2 is zero in the graph because it has a negative value, as the final concentration showed to be 55 mg/L, which is higher than the initial of 50 mg/L

##### **Conclusion:** From our metal-binding short peptides and proteins, the CSP1, MT-SYNSP, MymT and CSP1.3 had a better removal percentage than the pBAD empty backbone and the DH10B. Also, the synthetically modified CSP1.3 has a better removal than its original form.

### 4.2 Cobalt

For the cobalt colorimetric, we use a 50% potassium thiocyanate solution, which forms a blue cobalt complex and has the strongest absorbance at 620 nm [2].

![Standard curve for cobalt in cuvettes](https://static.igem.wiki/teams/5060/standard-curve-cobalt.png)

Figure 7: The standard curve of the cobalt colorimetric assay with deionized water with the cobalt concentrations of 0, 10, 25, 50, 100, 200, 250 and 300 mg/L

However, as we could not clone a cobalt-binding protein, we were unable to continue this experiment.

## 5. Polymerization

We observed clear fluorescence from both constructs, confirming successful expression of sfGFP and mCherry. After mixing the lysates containing the individual monomers, we detected **high molecular weight products**  on the SDS-PAGE gel, indicating that **polymerization had occurred.** This demonstrates that the system is capable of forming polymers through the interaction of SpyTags and SpyCatchers, validating the feasibility of using this approach for future applications, such as metal absorption.

![Picture of polymerisation result](https://static.igem.wiki/teams/5060/photo-2024-09-24-09-44-33.jpg)

![Fig. 8 SDS-PAGE of Tag-sfGFP-Tag and Catcher-mCherry-Catcher](https://static.igem.wiki/teams/5060/sds-page-t-sfgrp-t-and-c-mcherry-c.png)

Figure 8: SDS-PAGE of Tag-sfGFP-Tag and Catcher-mCherry-Catcher. Samples were prepared using two different temperatures. Lane 2 was scanned with different exposure to see the pattern at higher molecular weight.

Lane 1 ladder

Lane 2 TsfGFPT + C-RFP-C (37°C)

Lane 3 TsfGFPT (95°C)

Lane 4 C-RFP-C (95°C)

Lane 5 TsfGFPT + C-RFP-C (95°C)

Lane 6 pellet TsfGFPT (95°C)

Lane 7 pellet C-RFP-C (95°C)

Lane 8 TsfGFPT (37°C)

Lane 9 C-RFP-C (37°C)

### References

[1] Rocha, S. A. do N., Dantas, A. F., Jaeger, H. V., Costa, A. C. S., Leão, E. dos S., & Gonçalves, M. R. (2008). Spectrophotometric determination of copper in sugar cane spirit using biquinoline in the presence of ethanol and Triton X-100.  *Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 71*(4), 1414–1418. <https://doi.org/10.1016/j.saa.2008.04.013>

[2] NTU (n.d.).  *Quantitative analysis of cobalt (II) ions*   [https://teaching.ch.ntu.edu.tw/gclab/en/pdf/manual/10\_E10-M-Cobalt(II)-ions-2021.02.05.pdf](https://teaching.ch.ntu.edu.tw/gclab/en/pdf/manual/10_E10-M-Cobalt%28II%29-ions-2021.02.05.pdf)

{% endblock %}