Results

Summary

Cloning

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

Agar Plate

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

Toxicity

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

Colorimetric

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

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 to go to the Parts section.

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 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:

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 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.

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.

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.

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

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 to go to the Measurement section.

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.

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].

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.

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