iGEM 2023 — Team athens


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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Engineering

# Engineering Cycles

## Introduction

In this page, we break down our approach to designing and assembling the basic components of our project; the mutagenic M13 phages. Our methodology is anchored in engineering principles and follows the Engineering Design Cycle, comprising four key stages: Design, Build, Test, and Learn. The first engineering iteration begins with “Research”, and contains important information that justifies our further choices, laying the groundwork for subsequent phases.

Within these cycles, we highlight our multiple cloning attempts in order to get the desired engineered M13 phage. More specifically, we shed light on how we addressed project challenges, what we learned from each attempt, and the actions taken to improve our designs and results. At each stage of the cycles, you'll find a detailed description, including thoughts, results, and key decision points. These cycles, when examined in total, present our project through the prism of Engineering success, and highlight the logic before every team’s decision to finally achieve the desired M13 modification.

## ENGINEERING

* settings Created with Sketch Beta.

  [Cycle 1](#engineering-cycle1)
* settings Created with Sketch Beta.

  [Cycle 2](#engineering-cycle2)
* settings Created with Sketch Beta.

  [Cycle 3](#engineering-cycle3)

# Cycle 1

Research:

The main goal of our whole engineering process was to modify the M13 bacteriophage, isolate it and depose it on a gold-coated silicon wafer. This would enable the production of the patterns necessary for our Volatile Organic Compounds (VOC) to bind, leading to colorimetric output.

Specifically, we aimed to modify the phage’s pVIII or Major Coat Protein, which is expressed in approximately 2700 copies in the phage’s capsid. It is a 73 amino acid-long protein, although the first 23 amino acids are cleaved before finalization of the phage particle’s assembly, functioning as a signal peptide, leaving a 50 amino acid-long helical protein on the phages surface, with the N-terminal exposed on the surface. This outer protein region is the target of our modification, where we aimed to insert a short amino acid sequence exhibiting high binding affinity for our compounds. Thus, we first needed to discover these sequences.

Initially, phage display was considered for this task. However, limited laboratory experience, cost and time constraints prevented us from performing it, and we relied on in silico methods instead. More details on this can be found in our first engineering cycle as well as in the Dry Lab category of our wiki.

Once the desired sequences were found, we attempted to integrate them into the phage’s genome using the M13KE vector. For this purpose, we needed to explore our options. Initially, homologous recombination was considered, as it is a relatively easy method for phage genetic engineering. However, only by using this method only a limited amount of phages would potentially obtain the desired sequence, leading to a non-optimized (low engineered to non-engineered phage ratio) sensor [2]. Thus we were able to identify other methods involving mutagenic PCR and traditional cloning that are discussed both in the current page and in our Design page.

Design:

During the design phase, bioinformatic analyses were being conducted in order to determine the optimal amino acid sequences to be inserted into the M13 pVIII N-terminal region. For this purpose, a very large number of sequences were being generated and tested sequentially using Molecular Docking (see Model).

Wet lab experimental setup required the presence of an amino acid in position 1 whose codon starts with Guanine: Alanine, Valine, Aspartate, Glutamate or Glycine. This was to incorporate a PstI recognition sequence in the forward PCR primer, and enable the removal of the original sequence later down the line [1] (see Design). The sequence would also need to satisfy sensitivity (high binding affinity) and specificity (lower binding affinity for the other ligands) related conditions. A sequence satisfying the above was, at that point, DYYAW, chosen to bind with Hippuric Acid. Its coding sequence was incorporated into a PCR primer and prepared for ordering.

Forward: 5′-ATATATCTGCAGACTATTATGCGTGGCCCGCAAAAGCGGCCTTTAACTCCC-3′

Reverse: 5′-CACCCTCTGCAGCGAAAGACAGC-3′

Insert sequence in bold, PstI sequence underlined.

Before that, we decided to validate its efficacy in a manner that would more closely emulate real life conditions.

Build:

We inserted our chosen sequence (DYYAW) into the pVIII protein sequence at the appropriate position (position 2-6, replacing EGDD) and folded it using AlphaFold2. This would, in our opinion, constitute a better representation of the actual system. We prepared our protein and our ligand molecule appropriately and performed molecular docking simulations using Autodock Vina software.

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle1-image1.png)

Test:

The folded modified pVIII protein produced a much worse result than initially anticipated, with binding affinity scores dropping from -4.407 kcal/mol to -3.398 kcal/mol. This constituted an unacceptable drop in sensitivity for our potential future biosensor.

Learn:

We attributed this disappointing result to the presence of Alanine in position 1, right before the point of insertion. To validate our results, we also performed docking with the hexapeptide ADYYAW and found it to closely match the folded protein (-3.443).

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle1-image2.png)

![](https://static.igem.wiki/teams/4810/wiki/engineering/engineering90.png)

We concluded that we should continue testing hexapeptides, starting with Alanine and followed by a five amino-acid sequence satisfying the other conditions stated in the “Build” part of the cycle. Introducing a fixed Alanine would not affect the number of peptides we had to test, and would only minimally increase docking time for each receptor since the average search grid needed would be slightly greater.

# Cycle 2

Design:

Once the Dry lab concluded to one optimal sequence for each VOC, we had already designed the method to insert it in the M13 phage. In order to genetically modify the bacteriophage we used M13KE vector DNA

According to dry lab results M13KE DNA needed to be modified in order to incorporate the following genetic modifications in the gene of pVIII protein:

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle2-image1.png)

Literature research, provided us with PCR based protocols for M13KE phage engineering [1], which depended firstly on
site-directed mutagenesis PCR, in order to remove the unique PstI restriction enzyme recognition site of the vector,
and then on a second mutagenesis PCR in order to induce the desired modification in the pVIII gene. More information
about the selection and the design of these PCRs can be found in our [Design](https://2023.igem.wiki/athens/design) page.

The strategy is depicted in brief, graphically in Figure 1.

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle2-image2.png)

Build:

To obtain the mutagenic M13 phage, two PCR reactions were performed as described above. To verify their efficiency, 5μl samples
of each reaction were evaluated, by loading them on a 0.8% agarose gel (Figure 2). The DNA band of the linearized M13/ the product
of the PCR, was expected to be seen at ~7200 bp and thus between the second and third ladder band. However, no such bands were
observed and we came to the conclusion that the reaction was not successful.

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle2-image3.jpg)

Figure 2: DNA ladder 5μl b) 2μl PCR reaction

After our initial results several repetitions of the PCR reactions were performed, examined by electrophoresis. These attempts were preceded by constant evaluation, consultations with our advisors and our own research. During our troubleshooting various thermocycling conditions and reagent’s quantities and ratios were examined with no successful results. (examples can be seen in Figure 3). In brief, we altered the number of cycles, annealing and extension time and temperature, initial and normal denaturation time and reaction volume. We also tried to linearize the M13 genome before the reaction, add DMSO, preheat the primers and change the thermocycler we used.

![Image 1](https://static.igem.wiki/teams/4810/wiki/engineering/cycle2-image4.png)

![Image 2](https://static.igem.wiki/teams/4810/wiki/engineering/cycle2-image5.png)

Figure 3: Μultiple different PCR reactions showing no anticipated result. You can find the conditions and quantities that correspond to each sample in the “Lab Notebook”.

Learn:

The nature of our results, and more specifically the absence of clear bands, even of non-specific products, and the high frequency of smearing, indicated that the most probable cause of failure lies in the design of our primers given that we have performed extensive troubleshooting to pinpoint other potential causes [3]. After further investigation, we discovered that ΔGs’ absolute value of the formation of hetero-dimers was higher than values recommended by many primer designing tools. Thus, new codon choices of the mutagenic primers were made in order to minimize hairpin, homo and hetero dimer formation, using the IDT OligoAnalyzer tool.

Design:

In order to avoid wasting time, we made three different pairs of primers with slightly different sequences, length and GC content, to simultaneously test all three in order to theoretically have more chances for success. We designed partially complementary mutagenesis primers, with 1-3 non-complementary bases on their ends, to minimize dimer formation and improve the chances of primer-to-template binding.
All the newly designed primers had a lower tendency to form hetero-dimers and can be seen in “Design”.

Build (continue…)

To build upon the new design, further PCR reactions were attempted with the new sets of primers. Again many different cycling and mixing conditions were used. To verify reactions efficiency, 5μl samples of each reaction were evaluated, by loading them on a 0.8% agarose gel (Figure 4).

![Image 1](https://static.igem.wiki/teams/4810/wiki/engineering/cycle3-image1.png)

![Image 2](https://static.igem.wiki/teams/4810/wiki/engineering/cycle3-image2.png)

Figure 4: [Lab Notebook](https://2023.igem.wiki/athens/wet-lab-notebook) (example of PCR results numbered as 13’’-14’’-15’’-16’’)

Test:

Since the desired phage was not obtained with this technique, the “Test” step could not be performed in this
iteration of the cycle.

Learn:

Unfortunately, we still did not observe the desired product. We concluded that the specific PCR reaction to induce the desired mutation wasn’t as effective as we had initially anticipated. Thus without further ado, we agreed upon redesigning fundamentally. We changed our strategy to traditional cloning technique and discontinued the use of PCR mutagenesis to obtain our modified phage.

# Cycle 3

Design:

We considered inserting the desired sequence in the pVIII protein gene, by designing and ordering gBlocks/Gene Fragments. Specifically, the sequence would be incorporated in a larger insert fragment. This fragment, which has a different restriction enzyme site on each of its ends, corresponds to the original fragment of M13KE sequence, with the difference of a set of 15bp, that represents the desired modification.

We decided that the insert sequence would be cloned into the M13KE vector with double restriction, using KpnI and BsrGI enzymes. These fragments would have a length of 593bp.

In order to distinguish modified from unmodified M13KE DNA, a new HindIII restriction site was introduced, by inducing a silent mutation inside “M13 geneIII”. When digested with HindIII, the genetically engineered phage would produce two fragments (4649 bp and 2573 bp), instead of the one product of 7222 bp of the unmodified M13KE produced by single-site digestion.

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle4-image1.png)

The green arrow represents the designed gBlock/Gene Fragment sequence.

Build:

To acquire the wanted construct, we performed double digestions as described above, on both the M13KE vector and the insert.
The desired digested products were visualized through loading on an agarose gel (outlined on Figure 5), and then extracted
using a “Gel Extraction kit”. Through electrophoresis of the extracted product, clear bands were seen at ~600 bp and ~4500
bp respectively as expected (Figure 6).

![Image 1](https://static.igem.wiki/teams/4810/wiki/engineering/cycle4-image2.png)

Figure 5: Digestion products (A)

![Image 2](https://static.igem.wiki/teams/4810/wiki/engineering/cycle4-image3.jpg)

Figure 6: Gel extraction elutions

After obtaining the desired DNA fragments having the appropriate cohesive ends we proceeded to ligation reaction using a 1:2 and 1:3 molar vector:insert ratio.

Test:

In order to test if the cloning was successful, we proceeded to chemical transformation of XL1-blue cells with the ligation product.
A positive (PUC19 plasmid) and negative (only cells) transformation control were added, as well as a ligation negative control
(only digested vector, no insert). Some results of the first few attempts were the following:

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle4-image4.jpg)

Figure 7: All cultures showed phage growth, including the negative control. After examining the DNA sequences of a few plaques, by isolating it and digesting it with HindIII, we concluded that the plaques were the result of contamination from unmodified M13KE.

![](https://static.igem.wiki/teams/4810/wiki/engineering/cycle4-image5.jpg)

Figure 8: Only the positive control showed phage growth, meaning that the ligase reaction was probably unsuccessful.

Build:

In this iteration of cloning, it was imperative to rebuild by testing various different vector:insert ratios in the ligation process (for more detailed information check “Lab Notebook”).

Test:

![](https://static.igem.wiki/teams/4810/wiki/engineering/engineering40.jpg)

Figure 9: The growth was as expected at positive and negative controls. Eicosane and hippuric 1 sequences, plaques were also observed. This indicated that the engineered phage might be finally constructed.

From the results shown above, plaques - and thus hopefully modified phages - had occurred. The corresponding ligation reactions were the only ones where the molar vector:insert ratios were changed in favor of lower insert quantity, (to 2:1 and 3:1 respectively) showing that the ratios could be the source of the problem for our previously failed experiments.

![](https://static.igem.wiki/teams/4810/wiki/engineering/engineering41.jpg)

Figure 10: In this attempt, we used the molar ratio that gave us plaques in all of the ligase reactions and got plaques from all the sequences tested.

From the results shown above, plaques - and thus hopefully modified phages - had occurred. The corresponding ligation reactions were the only ones where the molar vector:insert ratios were changed in favor of lower insert quantity, (to 2:1 and 3:1 respectively) showing that the ratios could be the source of the problem for our previously failed experiments.
To ensure that the plaques were indeed formed from modified phages, we isolated DNA from a few of them and proceeded to digestion with HindIII.

Before digestion with HindIII samples were loaded in 1% agarose gel producing the following results:

![](https://static.igem.wiki/teams/4810/wiki/engineering/engineering45.png)

Most samples show three bands: 1) between 2kb and 3kb, 2)between 3kb and 4kb 3)between 6kb and 8kb corresponding to the circular M13 DNA.

Results after digestion with HindIII (the incubation time was induced due to lack of time)

![](https://static.igem.wiki/teams/4810/wiki/engineering/engineering51.png)

Most samples showed two to three zones: 1) between 2kb and 3kb 2) between 6kb and 8kb 3) at 4.5kb. Samples 4 and 13 show another unique band between 2kb and 3kb that could potentially correspond to one of the two fragments expected to result from HindIII digestion in two sites since two HindIII sites exist only in the modified bacteriophage.

Modified M13 DNA is expected to produce 2 bands at 4600 kb and 2600 kb when digested by HindIII. These bands are observed in the current electrophoresis in samples 4 and 13. It should be noted that the two bands did not coincide with the ones before HindIII digestion.

Therefore, highlighted bands serve as an indication of the possibility of having successfully modified the bacteriophage.

Learn:

From the above, we conclude that we have evidence of having genetically engineered M13 bacteriophages modified to bind with Hippuric Acid and Perillaldehyde.

In order to repeat and verify the results, an isolation-digestion procedure needs to be performed again but with longer incubation time, in order for the enzyme to produce more of the digested product. The electrophoresis will also last for a larger period of time, running at lower Voltage, in order to be able to have clearer bands. In parallel, the rest of the samples 4 and 13, will be sended for sequencing.

Restriction digestion with HindIII suggests that there is high probability that the genetically engineered M13 bacteriophage for the binding of Hippuric Acid and Perillaldehyde have been successfully constructed. Given that the success occurred in ligation mixtures with high vector/insert ratio we conclude that there is a strong probability of this ratio to be a significant factor for the success or failure of the ligation.

Improve/Future iteration’s suggestions:

Despite the construction of our M13 bacteriophage, our extremely limited time was prohibitive for further repeating the procedure for verification, amplification, purification and then testing of the modified M13 bacteriophage.

For the further development of our project, we would test the binding of the bacteriophage using Tryptophan Fluorescence Assay as described in our “Design” page. Results of the binding assay would be very informative for evaluating the effectivity of our molecular docking simulations and could serve as a guide for further optimizing amino acid selection, not only for the detection of the specific VOCs, but also for other compounds associated with diseases such as cancer [4].

Equally imperative to our project, is the test of the colorimetric output of the engineered bacteriophages when self-assembled on gold-substrates, which constitutes our end product. This test would serve as an evaluation of the effectiveness of the engineered M13 bacteriophage as a colorimetric-biosensor.

Nevertheless, despite the indication of having successfully engineered two types of M13 bacteriophages (for Hippuric Acid and Perillaldehyde binding) it should be noted that the modifications probably affected their viability. This can be derived by the fact that single plaques of the modified phages were observed with no prior dilution, something that is not common for M13 bacteriophage [5]. This suggests probable alternation of the viability of the bacteriophage, which could also serve as an explanation for failed transformations resulting from non-reproducible bacteriophage particles.

In order to explore the potential scenario of M13 phage particles losing their infectivity by the genetic modification of pVIII protein, we would need to redesign our strategy for choosing amino acid sequences that bind to the VOCs. Specifically, we would take into consideration physical and chemical alterations that could probably significantly interfere with the bacteriophage’s life cycle. For example, the electrical charge of the N-terminus of the pVIII protein could reduce the viability and/or infectivity of the particles [6]. That means that during a future bioinformatic analysis the charge of the amino acid sequence needs to be taken into consideration.

In conclusion, our engineering involved multiple iterations of the design-build-test-learn cycles, recruiting both wet and dry lab methods. Every engineering cycle served as a stepping stone for the development of the genetically engineered bacteriophages. The last engineering cycle ended with promising results of producing the engineered M13 phages designed to bind with Hippuric Acid and Perillic Aldehyde.

REFERENCES

* [1] - Lee, J., Warner, C., Jin, HE. et al. Production of tunable nanomaterials using hierarchically assembled bacteriophages. Nat Protoc 12, 1999–2013 (2017). https://doi.org/10.1038/nprot.2017.085
* [2],[5]-Mahler, M., Costa, A. R., Van Beljouw, S. P. B., Fineran, P. C., & Brouns, S. J. J. (2023). Approaches for bacteriophage genome engineering. Trends in Biotechnology, 41(5), 669–685. https://doi.org/10.1016/j.tibtech.2022.08.008
* [3] - Lorenz TC. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp. 2012 May 22;(63):e3998. doi: 10.3791/3998. PMID: 22664923; PMCID: PMC4846334.
* [4]- Janfaza S, Khorsand B, Nikkhah M, Zahiri J. Digging deeper into volatile organic compounds associated with cancer. Biol Methods Protoc. 2019 Nov 27;4(1):bpz014. doi: 10.1093/biomethods/bpz014. PMID: 32161807; PMCID: PMC6994028. (για recombination)
* [5] - Green MR, Sambrook J. Plating Bacteriophage M13. Cold Spring Harb Protoc. 2017 Oct 3;2017(10):pdb.prot093427. doi: 10.1101/pdb.prot093427. PMID: 28974654.
* [6]Iannolo, G., Minenkova, O., Petruzzelli, R., & Cesareni, G. (1995). Modifying Filamentous Phage Capsid: Limits in the Size of the Major Capsid Protein. Journal of Molecular Biology, 248(4), 835–844. doi: 10.1006/jmbi.1995.0264

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## Experiments

# Experiments

In this section, we present a comprehensive collection of the protocols we used during
our journey. These protocols reflect the core of our project and work as a brief, yet easy to understand, guide for synthetic
biology experiments. Taking into consideration the significance of well-documented processes, we tried our best to ensure that
they do not only represent our work but also serve as references for future teams. The protocols are optimized and interspersed
with notes based on our own experience for the organisms we used (M13 bacteriophage and XL1-blue bacteria). Please feel free to
explore and use these protocols as a resource and support for your own projects and experiments!

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## Results

# Aim

The aim of our project was to design genetically engineered bacteriophages able to bind
with specific volatile organic compounds (VOCs) found in altered concentration in
patients with Parkinson's disease. The binding is based on a modified peptide
sequence of the N-terminus of pVIII coat protein of bacteriophage.

#### For this purpose two important checkpoints had to be achieved:

1) The discovery of the specific peptides that bind with the specific VOC.
2) The construction and testing of the engineered bacteriophage.

In the current page results of both objectives are
presented uncovering milestones achieved towards the production of the diagnostic
test.

## RESULTS

* [Introduction](#introduction)
* [Data Analysis](#data-analysis)

## Construction and Test of the engineered bacteriophage

Multiple attempts utilizing different strategies to create the modified M13
bacteriophages were performed but unfortunately we didn't manage to isolate and
test the M13 bacteriophage particles.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results1.jpg)

Nevertheless after chemical transformation of E.coli cells with the ligation mix of the cloning reaction. Blue plaques
were observed and DNA was isolated. From the results of the restriction of the isolated DNA with HindIII we assume that we
have produced the modified M13 bacteriophage for the detection of Hippuric Acid and Perillaldehyde as seen below:

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results2.png)

Undigested isolated DNA from blue plaques of chemical transformation of ligation mix with E.coli cells.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results3.png)

Digested DNA with HindIII for confirmation of the modification of the bacteriophage.

Modified M13 DNA is expected to produce 2 bands at 4600 kb and 2600 kb when digested by HindIII. These bands are observed in the current electrophoresis in samples 4 (for Hippuric Acid) and 13 (for Perillaldehyde). It should be noted that the two bands did not coincide with the ones before HindIII digestion. Therefore, highlighted bands serve as an indication of the possibility of having successfully modified the bacteriophage since the highlighted bands seem to correspond to the 2600bp fragment produced by HindIII restriction while the other fragment of size of 4600bp is also present.

## Data Analysis

#### Targeting VOC Specificity:

Our primary objective was to identify peptides with a high binding affinity for specific volatile organic compounds (VOCs) while maintaining low affinity for other non-target VOCs. In our pursuit of achieving this, we focused on finding peptides that possessed three critical characteristics:

##### 1. High Affinity Score:

We sought peptides with a high affinity score towards our target VOC, ensuring effective binding.

##### 2. Selectivity:

It was imperative for these peptides to exhibit selectivity when compared to other VOCs of interest.

##### 3. Discrimination from unrelated compounds:

To distinguish the target VOC from other compounds, we emphasized the need for peptides with remarkable discrimination abilities.

Thus, we considered the presence of other VOCs commonly found in human sebum, making the task more intricate.

#### Iterative Search Process:

Transitioning from tripeptides to tetrapeptides, we embarked on a labor-intensive quest. With countless potential tetrapeptides, we had to devise a strategic approach. Given the extensive computation time required to analyze all tetrapeptides with our ligands, and our relatively low computational power, we decided to employ targeted searches based on patterns emerging from previously tested peptides. Using Python code, we identified trends, such as an overrepresentation of tryptophan (W) in eicosane results, and adjusted our strategy accordingly.

In addition to these criteria, we set specific "rules" for our target peptide:

##### 1) It should initiate with Alanine.

##### 2) The second position should contain A, V, D, E, or G.

Notably, we included Alanine as the first amino acid to mimic the cleaved pVIII protein realistically. We proceeded to explore pentapeptides and hexapeptides in our search, generating hexapeptide derivatives based on our top-performing tetrapeptides. Nevertheless, no significant correlations were found between tetrapeptides and their hexapeptide derivatives, necessitating the generation of random hexapeptides for further analysis.

#### Building the Peptide-VOC Database:

With extensive experimentation and filtering, we organized our results into a comprehensive peptide-VOC binding affinity database, consisting of a staggering 298,711 unique peptides. More information about it can be found on our [Software](https://2023.igem.wiki/athens/software) page.

#### Data Analysis and Optimization:

To maximize the utility of this database, we conducted a series of data analyses:

##### Binding Affinity Distribution:

We plotted the binding affinities for each VOC and calculated the mean, providing insights into the overall binding characteristics.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results4.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results5.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results6.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results7.png)

##### Amino Acid Favoritism:

Investigating the top 10% of peptides for each VOC, we sought clues about amino acid preferences.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results8.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results9.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results10.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results11.png)

##### Optimal Peptide Length:

We determined the peptide length group (from tripeptides to heptapeptides) with the highest absolute binding affinity.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results12.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results13.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results14.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results15.png)

##### VOC Ranking:

We evaluated which of the four VOCs exhibited the highest absolute mean binding affinity with the tested peptides.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results16.png)

##### Hydropathicity Correlation:

Exploring the correlation between hydropathicity (using the Kyte-Doolittle scale) and binding affinity, we aimed to uncover any relationships, but found that the Pearson correlation was consistently close to zero for all four VOCs.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results17.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results18.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results19.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results20.png)

##### Experimental Validation:

Having distilled our results, we selected the most promising peptides for each VOC and examined their binding affinity with the remaining three VOCs. Subsequently, we rigorously tested the 4 selected peptides with a total of 22 common skin volatiles.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results21.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results22.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results23.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results24.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results25.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results26.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results27.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results28.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results29.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results30.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results31.png)

We went a step further by modifying the protein through the incorporation of the selected peptides, followed by testing
the modified proteins against the wild type, providing valuable insights into the practical implications of our findings.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results32.png)

This comprehensive approach allowed us to navigate the complex landscape of peptide-VOC interactions and optimize the selection
of peptides for a wide range of practical applications.

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results33.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results34.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results35.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results36.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results37.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results38.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results39.png)

![results-image](https://static.igem.wiki/teams/4810/wiki/results/results40.png)

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