iGEM 2025 — Team ulaval

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# Notebook

![Barre](https://static.igem.wiki/teams/5653/education/barre.webp)

This page presents the full timeline of our **Totally [Fe]rocious** project. Our journey began with the recruitment of new members in November 2024. By January, once everyone was integrated, we started brainstorming project ideas and conducting extensive literature research to define our objectives for the year. All year long, we reached out to field experts to refine our goals based on identified knowledge gaps. We also initiated fundraising efforts and planned our education and communication events for the year. Throughout the summer, both our wet lab and dry lab teams worked intensively to advance the project.

1. November 2024
2. Nov 1

   #### Recruitment starts

   Starting the recruitment for the next iGEM season, send acceptance emails to new team members until January 31st.
3. December 2024
4. Dec 3

   #### First team meeting

   Integrated new members to the team & introduction to the iGEM competition.
5. January 2025
6. Jan 14–30

   #### Brainstorm & literature review

   Research and brainstorming sessions on project ideas.
7. Jan 30

   #### Project ideas shortlist

   Selection and elaboration of project ideas.
8. Jan 31

   #### Recruiting period ends

   Active recruiting period stopped, welcome to the new and returning iGEM members!
9. February 2025
10. Feb 1

    #### BVE grant form

    Wrote the *Bureau de la vie étudiante* grant form.
11. Feb 3

    #### Team registration
12. Feb 6

    #### Final idea selection

    Final selection of project ideas: we must now make our choice.
13. Feb 13

    #### Project topic chosen Antibiotic resistance

    We began doing more elaborate background research on this subject.
14. March 2025
15. Mar 14–16

    #### Expo-sciences

    Participated in the 2025 regional finales of the Expo-sciences alongside the *Faculté des sciences et de génie* of *Université Laval*.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-expo-sciences.webp)
16. Mar 16

    #### BVE grant submitted

    The *Bureau de la vie étudiante (BVE)* grant form is submitted.
17. Mar 21–23

    #### *Rendez-vous en Sciences*

    Presentation of the iGEM competition, past projects, and this year’s project.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-rdv-en-sciences.webp)
18. Mar 25

    #### GoFundMe launched

    Crowdfunding campaign kickoff.
19. April 2025
20. Apr 15

    #### Name & logo finalized

    Final selection of the project name and logo: **Totally [Fe]rocious**.
21. Apr 17

    #### BVE Gala

    Participation and outreach at the *Bureau de la vie étudiante*.

    ![iGEM2025](https://static.igem.wiki/teams/5653/notebook/bve-igem2024.webp)
22. Apr 29

    #### Education activities

    Start of the creation of interactive activities for senior residences.
23. May 2025
24. May 1

    #### Plasmid & gBlocks

    Designed plasmid and gBlocks sequences.
25. May 5

    #### Team merch ordered

    iGEM ULaval merch order placed.
26. May 6

    #### Lab planning

    Planification of the Wet & dry lab activities.
27. May 14

    #### Safety Form v1

    Completed the first version of the Safety Form.
28. May 20

    #### Orders placed

    gBlocks (IDT) & PCR primers ordered.
29. May 21

    #### Safety Form finalization

    Revisions finalized.
30. May 21

    #### Human Practices

    Met with Dr. Patrick Laguë (expert in structural and dynamics properties in proteins and biological membranes).
31. May 29

    #### Human Practices

    Met with Dr. Élodie Boisselier (expert on gold nanoparticles in the retina).
32. May 30

    #### Wet lab works begins
33. June 2025
34. Jun 13–15

    #### *Festival Lévisium* kiosk

    Animated a vulgarisation kiosk on iGEM & this year’s project.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-levisium.webp)
35. Jun 17

    #### Promo video

    Began brainstorming for this year's promotional video and selected our Village.
36. Jun 19

    #### *Classique de Golf*

    Presented past iGEM projects with a special focus on this year's project.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-golf.webp)
37. July 2025
38. Jul 2

    #### Wiki writing

    Began initial structure and content drafting.
39. Jul 3, 15 & 29

    #### Senior residences outreach

    Introduced seniors in nursing homes to synthetic biology & our project.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-cogir.webp)
40. Jul 13

    #### Team photos & wiki meeting

    Took individual team photos and held our first in-person meeting to write the wiki pages.
41. Jul 20

    #### Promotional video filming

    First day of filming completed.
42. August 2025
43. Aug 3, 9, 10, 16, 17

    #### Wiki sprints

    Multiple in-person meeting to continue writing the wiki.
44. Aug 5

    #### Promo video done

    Filming & editing completed.
45. Aug 6

    #### Safety Form final rewrite

    Final polishing and checks.
46. Aug 20

    #### Check-in form submitted

    Deadline met.
47. Aug 22

    #### IBIS Student Day

    Presented iGEM and this year’s project at the *Journée étudiante de l'IBIS*.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-ibis.webp)
48. Aug 28

    #### Wet lab completed

    End of wet-lab work.
49. Aug 29

    #### FSG Welcome Day

    Presented our project during the *Journée d’accueil* at the *Faculté des sciences et de génie* of *Université Laval*.

    ![iGEM2025](https://static.igem.wiki/teams/5653/education/logo-fsg.webp)
50. Aug 30

    #### North Canadian Mini Jamboree

    Team participation.
51. September 2025
52. Sep 1

    #### Wiki code starts

    Initial implementation.
53. Sep 17

    #### Human Practices

    Met with Mireille Quémener, Ms.c, Ing (expert in optics and laser for biomedical engineering).
54. Sep 25

    #### Human Practices

    Met with Vanessa Fillion and Dr. Jeannot Dumaresq, healthcare manager and infectiologist (expert in the growing field of antibiotic resistance and infection).
55. October 2025
56. Oct 1

    #### Preliminary judging Title & abstract due

    Milestone submitted.
57. Oct 8

    #### Dry lab wrap-up

    Final results consolidated of the Dry lab.
58. Oct 8

    #### Wiki Freeze

    Content locked.
59. Oct 28–31

    #### Grand Jamboree

    Team attending.

    ![iGEM2025](https://static.igem.wiki/teams/5653/membersrealpictures/igemulaval-grandjamboree.webp)

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# Engineering success

![Barre](https://static.igem.wiki/teams/5653/contribution/barre.webp)

## Context

Totally [Fe]rocious's main goal was to stealthily kill *Klebsiella pneumoniae* using the siderophore aerobactin coupled to a gold nanoparticle.
We decided to synthesize aerobactin in *Escherichia coli* and planned to use the conjugated aerobactin in a limited iron
environment —to mimic an infectious site— and activate the complex ¨Fe3+ - aerobactin -AuNPs with a near-infrared (NIR) laser. To achieve this goal, we had multiple aims for the Wet and Dry
lab parts using the Design-Build-Test-Learn (DBTL) cycle. For the Wet lab section, we aimed to amplify, assemble
and synthesize aerobactin using an optimized *E. coli* strain before binding the AuNPs to the aerobactin to create our Trojan Horse.
As for the Dry lab, our goal was to gain a deeper understanding of the siderophore internalisation mechanisms in order to ensure that the
incorporation of an additional foreign moiety into the siderophore would not disrupt its uptake. This enhanced understanding could also help optimize
the “Trojan Horse” strategy and facilitate the efficient delivery of modified siderophores into bacteria.

## Design

**Wet lab**

The foundation of Totally [Fe]rocious was built upon the aerobactin gene: *iucA* to *D* from *K. pneumoniae's* siderophore aerobactin. It is a siderophore used by many bacteria. These genes were used to recreate the biosynthesis pathways of aerobactin in an optimized *E. coli* strain DH5𝞪. These genes, the *iucA* Citryl transferase, the *iucB* N⁶-acetyltransferase, the *iucC* Citryl transferase and the *iucD* L-lysine N⁶-monooxygenase have the potential to recreate the exact, and recognizable, siderophore in another bacteria before binding it with gold nanoparticles and destroying them from the inside-out.

Aerobactin from *K. pneumoniae* is a hydroxamate-type siderophore and can also be considered as a “xenosiderophore” because of its capability of being accepted as an active siderophore by other bacteria.

Considering the difficulty and limitations of our project, we set out to use *E. coli* DH5𝞪 before thinking of using *K. pneumoniae*. In that regard, we optimized each gene part to be in another bacteria than its original one. We also needed a way to observe if the construction that we have made was expressed or present in the DH5𝞪. For this, we decided to use the plasmid pBluescript a vector for our Gibson Assembly. This means that our aerobactin pathway was assembled in the pBluescript plasmid before we transformed our DH5𝞪 to express one of each of both colours: blue or white in the presence of IPTG in the petri dish.

## Plasmid construction

The *iucA* gene from *K. pneumoniae* has a length of 1395bp and initiates the core biosynthesis pathway of aerobactin. It requires citrate to be able to ligate this product as a first molecule (see the contribution/parts section or [BBa\_256Z6XQY](https://registry.igem.org/parts/bba-256z6xqy)). We designed the *iucA* gene with a TAC promoter, a random sequence and an RBS (Bba\_0029) upstream of the start codon of *iucA*.

The *iucB* gene from *K. pneumoniae* has a length of 945bp and performs the second and final enzymatic step in the biosynthesis (see the contribution/parts section or [BBa\_253NP2T0](https://registry.igem.org/parts/bba-253np2t0)). We designed the *iucB* gene with a spacer between the *iucA* and *iucB* genes, plus an RBS (Bba\_B0030) upstream of its sequence.

The *iucC* gene from *K. pneumoniae* has a length of 1779bp and acts as the second citryl transferase after *iucA*. It also carries the final biochemical reaction in the biosynthesis pathway (see the contribution/parts section or [BBa\_25TEHQLR](https://registry.igem.org/parts/bba-25tehqlr)). We designed the *iucC*> sequence with a spacer between *iucB* and *iucC* with an RBS (Bba\_B0032) upstream of the gene sequence.

The *iucD* gene from *K. pneumoniae* has a length of 1332bp and catalyzes the first step in the biosynthesis of ahLys (see the contribution/parts section or [BBa\_255DLKJB](https://registry.igem.org/parts/bba-255dlkjb)). We designed the *iucD* sequence with a spacer between *iucC* and *iucD* with an RBS (Bba\_B0064) upstream of the sequence, and put a terminator at the end with a random sequence.

With all these parts, we obtained the reference sequence that is the IucABCD-assembly: [BBa\_25ZGK3Y5](https://registry.igem.org/parts/bba-25zgk3y5).

**Dry lab**

## Genome Scale Metabolic Model (GEM)

To optimize the in-silico production of aerobactin, our first design objective was to identify the environment and genetic conditions that maximize intracellular **citrate availability** - the initial building block of our siderophore. We selected *E. coli* as the chassis for heterologous expression of its well characterized metabolism and availability of validated genome-scale models.

We used the **iML1515** model, which includes 1516 genes and over 2700 metabolic reactions, as the foundation for our simulations. Our design variables included:

* Carbon sources (*glucose, acetate, glycerol, succinate*),
* pH and temperature ranges,
* Nitrogen source (*NH4+* or inorganic phosphate),
* knockout of key reactions such as **ICDHyr** (isocitrate dehydrogenase)[1].

We hypothesized that low pH, alternative carbon sources, and specific knockouts could increase citrate accumulation at the expense of biomass growth, a desired trade-off for metabolic overproduction.

## Protein Sequences & Structure

At the core of this project lies the set of protein sequences essential to address our research
questions. Most notably, these include the four proteins responsible for siderophore biosynthesis,
as well as the sequences of the TBDT and TonB. However, limited information was available in the
literature regarding siderophore-associated proteins in *K. pneumonia*. Our initial
objective was therefore to gather as much structural information as possible, particularly on the
TBDT, to ensure that our prediction closely reflects the naive configuration observed in nature.

To achieve this, one of our first steps was to translate the extensive knowledge available for
*E. coli* to our organism of interest. Leveraging the high structural conservation of
TBDTs and other related proteins, we used BLASTp [2] searches restricted to *K. pneumonia*
(taxid:573) to identify homologous sequences. As for aerobactin, our siderophore of interest,
its Simplified Molecular Input Line Entry System (SMILE) code was available on PubChem [3].
Additionally, we sought information on potential post-translational modifications (PTMs), disulfide bridges,
and functional domains within the TBDT to provide key insights for subsequent analyses throughout the iGEM
project cycle.

## Biomodel Composition

In order to model the TBDT within a bacterial membrane, several additional questions needed to be
addressed to ensure the accuracy of the system. Having already identified the TBDT’s sequence and
structure, the next considerations concerned the composition of the bacterial membrane and the
surrounding solution. Facing the same challenge of limited information available for
*K. pneumonia*, we opted to use a generic Gram-negative membrane composition [4].
For its part, the solution was supplemented with Na+ and Cl- ions, as these are among the most
abundant ions found in biological environments. Finally, the CHARMM-GUI [5] platform was selected
to construct our systems, given its extensive set of tools and flexibility for various molecular
dynamic modelling approaches.

## Build

**Wet lab**

**1. pBluescript “backbone” Extraction [6]:** We used an extraction technique to extract the pBluescript plasmid from a liquid bacterial culture using the Quiagen kit: QUIAprep spin miniprep kit.

**2. pBluescript Digestion and Linearization [6]:** We used the HindIII-HF enzyme to digest the plasmid and obtain its linear version that we eventually used for the Gibson Assembly.

**3. pBluescript Cleaning [6]:** After the digestion with the HindIII-HF enzyme, we cleaned up the DNA with the “BioBasic: EZ-10 Spin Column DNA Cleanup Miniprep Kit” to purify the DNA and remove unwanted components (contaminants).

**4. pBluescript Electrophoresis [6]:** After the clean-up, we put the DNA on a 0.8% agarose gel to observe if we had isolated the pBluescript plasmid in its linear form.

**5. Oligo Resuspension:** We resuspended our gBlocks oligos, along with their forward and reverse primers, to a specific concentration.

**6. gBlock PCR Amplification [6.a]:** We then used our resuspended oligos and amplified our gBlocks to add the homology arms necessary for Gibson assembly.

**7. PCR Electrophoresis [6]:** Using the same agarose gel recipe from before, we put our gBlock amplification on the gel to see if it had worked.

**8. PCR Touchdown:** We needed to upgrade our specificity for the third gBlock, to achieve that, we tried the PCR Touchdown method. This method works by having a higher temperature in the first 9 cycles before dropping at a rate of minus 1 degree Celsius per cycle all the way down to the primers' optimal annealing temperature.

**9. Gibson Assembly [6.b]:** Our goal was to assemble all of our gBlocks in the pBluescript plasmid, and for that, we used the Gibson assembly kit and method.

**10. Chemical Transformation of Competent Cells [6.c]:** Using the *E. coli*
strain DH5α, we transformed our competent cells with the Gibson assembly product before putting our transformation product on a petri dish culture. After 24h at 37℃, the clones that worked were white, and those that didn’t transform were blue (from the pBluescript plasmid).

**11. Plasmid Extraction [6]:** We extracted the plasmid from positive mutants using the “QIAgen - QIAprep spin miniprep kit” to be digested afterward.

**12. Digestion of Assembled Plasmid [6]:** After the plasmid extraction, we digested our plasmid construction to see if the Gibson assembly worked, using the BamI-HF and the EcoRI enzymes.

**13. Subsequent Amplification:** Before proceeding to the next step of our project, we needed the Gibson assembly to work, and for that, we needed the third gBlock to amplify in PCR. We then tried the inverse touchdown PCR method on the gBlock and the stitching PCR method.

**14. Competent Cells [6.c]:** After several cycles of troubleshooting, we finally tried to make our own competent cells to then assemble our gBlocks.

**Dry lab**

## GEM

The computational model was built and parameterized using the **COBRApy** Python package, which enables constraint-based reconstruction and analysis of genome-scale networks. We implemented flux balance analysis (FBA) to calculate steady-state flux distributions maximising either growth or citrate export to assess pathway flexibility [7].

We simulated multiple conditions by systematically varying media composition and environmental parameters, applying gene knockouts, and recording resulting fluxes for:

* Specific growth rate (h⁻¹),
* Citrate export flux (mmol/gDW/h),
* Siderophore secretion rate (mmol/gDW/h),

## Structure Predictions

Prediction of the siderophore aerobactin was performed using Chai-1 [8], which accepts SMILEs strings as valid ligand inputs. The TBDT structure was predicted using both the Chai-1 and Alphafold3 [9] servers. Since both accept the one letter code of amino acid, it permitted us to assert a sort of consensus on the resulting prediction. Residue-level predictions of membrane embedding were obtained using the OPM web server [10].

## Molecular Dynamics

Using the information gathered during the Design phase, along with the predicted 3D structure of TBDT, we constructed a model representing the in vivo composition of a Gram-negative bacteria membrane with the TBDT embedded at its core. The system was further surrounded by a sufficiently large water box to minimize potential interaction artifacts and supplemented with the appropriate ions. CHARMM-GUI provided the system, its parameters, and the optimized force field, enabling a high-quality and physically realistic model for subsequent simulations.

## Test

**Wet lab**

**\*From this point forward, the test and learn section for the wet lab are hypotheses about what the next steps should be about and what we could learn from them.\***

With our assembled plasmid carrying the three gBlocks, and within them all the necessary genes to produce and export the aerobactin siderophore, the next step would be to put the bacterial strain in a rich citrate environment to induce the production of aerobactin. Within the culture, we would need to detect the presence of siderophores using Mass-Spectrometry (MS) and other chemical tests for the hydroxamate siderophore-type, like the *Csaky Test* that depends on the formation of NO2- via the oxidation of hydroxylamine (NH2OH) by an iodine solution and the formation of colored dye, the *Ferric Perchlorate Assay* that identifies, by the appearance of an orange-red or purple color, the hydroxamate-type siderophores when putting Fe3+ ion and low pH perchloric acid in the test tube, or the *Tetrazolium Test* that only uses a few drops of NaOH and the sample to the tetrazolium salt creating a deep-red color [11]. Eventually, we would need to conjugate our siderophore to a gold nanoparticle to try and find a way to activate the complex aerobactin-Fe3+-AuNP inside *K. pneumoniae*.

**Dry lab**

### GEM

Simulations showed that **maximum citrate secretion (18.17 mmol/gDW/h)** occurred under acidic conditions (pH ≈ 5) with *glycerol* as the carbon source and *Pi* as the nitrogen source. In contrast, the wild-type (WT) model consistently achieved the highest growth rate (~1.85 h⁻¹) under neutral pH with ammonium, confirming a clear metabolic trade-off between citrate production and biomass formation.

### Molecular Dynamics

We used NAMD [11] to simulate three independent replicates of the TBDT system with the domain, as well as a second set of three replicates without the plug domain, each for a minimum duration of 350 ns for each trajectories. Upon completion of the simulations, the membrane systems trajectories were visualized using VMD (Visual Molecular Dynamics) [13]. Finally, most computational analyses were performed using Wordom [14].

## Learn

**Wet and Dry lab**

Our Wet lab experiments were cut short because of our inability to assemble our three gBlocks into a functional plasmid. However, the entire sequence should be revised to know why the third gBlock is so difficult to amplify before trying any PCR methods. We suspect having some more difficulties with the expression of the siderophore and the conjugation with the AuNPs, but nothing unachievable. The expected results should be the successful binding of the siderophore-AuNPs and the formation of the aerobactin-Fe3+-AuNP complex that will use the TBDT-TonB system to enter *K. pneumoniae*. After this step, we should be able to activate the complex with a NIR laser (808nm) that would be absorbed by both AuNPs and *K. pneumoniae*. The NIR laser would activate the conjugate AuNPs that will, then, vibrate, produce heat and induce cellular lysis.

By then, we suppose that we will have learned if the use of a NIR laser is effective or if another method should be used, whether the AuNPs are optimal for the binding and whether the lysis effect is the one that the Totally [Fe]rocious team aimed for.

### GEM

From this first modelling iteration, we learned that citrate accumulation can be enhanced in acidic, glycerol-rich environments but at the cost of growth. This insight indicates that  **two-stage fermentation strategy**: first growing cells in neutral conditions, then switching to acidic media for production would likely optimize total aerobactin yield. We also learned that maintaining siderophore flux despite *ICDHyr* deletion suggests possible redundancy in the citrate synthesis routes, warranting further exploration via flux variability analysis and double knockouts.

### Molecular Dynamics

The dry lab investigation of the plug domain’s contribution to the TBDT structure provided valuable insights at the residue level, clarifying how its presence influences the β-barrel and overall protein stability. However, a more rigorous experimental design could have yielded a stronger dataset to analyse. Initiating the calculation earlier on, for instance, would have enabled the generation of longer trajectories, allowing for the observation of more stable protein conformations, even within the limited timeframe of an iGEM cycle. Similarly, increasing the number of replicates to five would provide us a clearer picture of the interactions governing the TBDT dynamics and its plug domain.

The scope of our research was also constrained by the absence of parametrization for the siderophore-Fe3+ complex. Future work could focus on developing optimized parameters for various siderophores in complexes with Fe3+, enabling the study of a broader range of bacterial systems. Incorporating these newly parametrized complexes into simulations would provide a deeper insight into the molecular interactions governing siderophore translocation through the TBDT. Ultimately, this would improve our understanding of the siderophore transport across the outer membrane and could extend to characterizing their crossing through the inner membrane, across an ABC transporter.

## References

1. Monk, J. M., Lloyd, C. J., Brunk, E., Mih, N., Sastry, A., King, Z., Takeuchi, R., Nomura, W., Zhang, Z., Mori, H., Feist, A. M., & Palsson, B. O. (2017). iML1515, a knowledgebase that computes *E. coli* traits. *Nature Biotechnology, 35*(10), 904-908
   <https://doi.org/10.1038/nbt.3956>
2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. *Journal of molecular biology, 215*(3), 403-410.
    [https://doi.org/10.1016/s0022-2836(05)80360-2](https://doi.org/10.1016/s0022-2836%2805%2980360-2)
3. Kim, S., Chen, J., Cheng, T., Gindulyte, A., He, J., He, S., Li, Q., Shoemaker, B. A., Thiessen, P. A., Yu, B., Zaslavsky, L., Zhang, J., & Bolton, E. E. (2025). PubChem 2025 update. *Nucleic Acids Res., 53*(D1), D1516–D1525.
   <https://doi.org/10.1093/nar/gkae1059>
4. Pogozheva, I. D., Armstrong, G. A., Kong, L., Hartnagel, T. J., Carpino, C. A., Gee, S. E., Picarello, D. M., Rubin, A. S., Lee, J., Park, S., Lomize, A. L., & Im, W. (2022). Comparative molecular dynamics simulation studies of realistic eukaryotic, prokaryotic, and archaeal membranes. *Journal of chemical information and modeling, 62*(4), 1036-1051
   <https://doi.org/10.1021/acs.jcim.1c01514>
5. Jo, S., Kim, T., Iyer, V. G., & Im, W. (2008). CHARMM-GUI: a web-based graphical user interface for CHARMM. *Journal of computational chemistry, 29*(11), 1859-1865
   <https://doi.org/10.1002/jcc.20945>
6. Protocol based on the Labarchives protocol for the Laboratoire de génétique moléculaire et de biologie synthétique course, W25, BCM-3010, Biochemistry, microbiology and bio-informatics department, Science and Engineering Faculty, Laval University, Québec, Canada. Annotated by Cyril Fraser-Laflamme. Original from the manufacturer Qiagen: QIAprep spin miniprep kit.
   1. Protocol based on the manufacturer’s (NEB) : PCR Using Q5®Hot Start High-Fidelity DNA Polymerase (M0493)
   2. Protocol based on the manufacturer’s (NEB) : Gibson Assembly® Protocol (E5510) | NEB
   3. Protocol based on the manufacturer’s (NEB) : Gibson Assembly® Chemical Transformation Protocol (E5510) | NEB
7. Orth, J. D., Thiele, I., & Palsson, B. Ø. (2010). What is flux balance analysis ? *Nature Biotechnology, 28*(3), 245-248.
    [https://doi.org/10.1038/nbt.1614](https://doi.org/10.1038/nbt.1614%20)
8. Boitreaud, J., Dent, J., McPartlon, M., Meier, J., Reis, V., Rogozhnikov, A., & Wu, K. (2024). Chai-1: Decoding the molecular interactions of life. *BioRxiv.*
   <https://doi.org/10.1101/2024.10.10.615955>
9. Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., ... & Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. *Nature, 630*(8016), 493-500.
   <https://doi.org/10.1038/s41586-024-07487-w>
10. Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I., & Lomize, A. L. (2012). OPM database and PPM web server: resources for positioning of proteins in membranes. *Nucleic acids research, 40*(D1), D370-D376.
    <https://doi.org/10.1093/nar/gkr703>
11. Gomes, A. F. R., Sousa, E., & Resende, D. I. S. P. (2024). A Practical Toolkit for the Detection, Isolation, Quantification, and Characterization of Siderophores and Metallophores in Microorganisms. ACS omega, 9(25), 26863–26877.
    <https://doi.org/10.1021/acsomega.4c03042>
12. Phillips, J. C., Hardy, D. J., Maia, J. D., Stone, J. E., Ribeiro, J. V., Bernardi, R. C., ... & Tajkhorshid, E. (2020). Scalable molecular dynamics on CPU and GPU architectures with NAMD. *The Journal of chemical physics, 153*(4).
    <https://doi.org/10.1063/5.0014475>
13. Humphrey, W., Dalke, A., & Schulten, K. (1996). VMD: visual molecular dynamics. *Journal of molecular graphics, 14*(1), 33-38.
    [https://doi.org/10.1016/0263-7855(96)00018-5](https://doi.org/10.1016/0263-7855%2896%2900018-5)
14. Seeber, M., Felline, A., Raimondi, F., Muff, S., Friedman, R., Rao, F., Caflisch, A., & Fanelli, F. (2011). Wordom: a user-friendly program for the analysis of molecular structures, trajectories, and free energy surfaces. *Journal of computational chemistry, 32*(6), 1183-1194.
    <https://doi.org/10.1002/jcc.21688>

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### On this page

# Experiments

![Barre](https://static.igem.wiki/teams/5653/education/barre.webp)

Here you will find our protocols, which cover bacterial expression to DNA assembly of our siderophore,
as well as troubleshooting steps. Each protocol contains all the materials needed for the experiment.
In addition, all steps are described so that future iGEM participants can effectively build upon our work.

In order to optimize the amount of DNA for our gBlock 3, the modifications made to the standard PCR
protocol are already described in the gBlock amplification protocol (PCR amplification) with the necessary
modifications to carry out the experiment. If you want to know more about this particular step, don’t forget
to look at our **[Results](https://2025.igem.wiki/ulaval/results)** page!

The vector assembly, pBluescript SK- with our 3 gBlocks, was assembled based on the manufacturer’s (NEB)
Gibson Assembly® Protocol (E5510) | NEB.

![Gibson assembly](https://static.igem.wiki/teams/5653/experiments/neb-gibsonassemblyworkflow.webp)

**Figure 1:**NEB Gibson assembly workflow [1]

We used the SOC medium (Super Optimal broth with Catabolite repression) to optimize the recovery of our
*Escherichia coli* competent cells following transformation. In the end, our mutant selection was effectively
done on LB agar plates with ampicillin, supplemented with X-gal, which allowed us to do a blue-white selection
(Gibson assembly & transformation - Chemical transformation of competent cells). **The cells that integrated our
vector appeared white.**

---

## Primers

Primers ordered from IDT. The primers were resuspended according to the manufacturer's instructions.

* Primers

  If the PDF does not display, [open it in a new tab](https://static.igem.wiki/teams/5653/experiments/primers-idt.pdf).

## Electrophoresis recipes

Recipes and buffers used for electrophoresis.

* Agarose gel (0.8%) recipe

  If the PDF does not display, [open it in a new tab](https://static.igem.wiki/teams/5653/experiments/agarose-gel-recipe.pdf).
* TAE (50X) buffer recipe

  If the PDF does not display, [open it in a new tab](https://static.igem.wiki/teams/5653/experiments/tae-recipe.pdf).

## Bacterial culture medium

Recipes for bacterial culture media used in our project.

* LB agar-ampicillin medium recipe

  If the PDF does not display, [open it in a new tab](https://static.igem.wiki/teams/5653/experiments/lb-recipe.pdf).
* SOB medium recipe

  If the PDF does not display, [open it in a new tab](https://static.igem.wiki/teams/5653/experiments/sob-recipe.pdf)
* SOC medium recipe

  If the PDF does not display, [open it in a new tab](https://static.igem.wiki/teams/5653/experiments/soc-recipe.pdf)

## Plasmid backbone extraction

Extraction, digestion, clean-up, and control steps.

* Extraction of pBluescript

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/pbluescrip-backbone-extraction.pdf)
* Digestion of pBluescript

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/digestion-of-pbluescript.pdf)
* Clean-up of pBluescript

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/pbluescript-clean.pdf)
* Electrophoresis

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/pbluescript-electrophoresis.pdf)

## gBlock amplification

Resuspension, PCR, and gel verification.

* Resuspension of oligos

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/oligo-resuspension.pdf)
* PCR amplification

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/gblock-pcr-amplification.pdf)
* Electrophoresis

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/pcr-electrophoresis.pdf)

## Gibson assembly & transformation

Gibson assembly and chemical transformation.

* Gibson assembly

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/gibson-assembly.pdf)
* Chemical transformation of competent cells

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/chemical-transformation-of-competent-cells-docx.pdf)

## Mutant confirmation

Plasmid extraction and verification digestion.

* Plasmid extraction from positive mutant

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/plasmid-extraction-from-positive-mutant.pdf)
* Plasmid digestion

  [Ouvrir le PDF](https://static.igem.wiki/teams/5653/experiments/digestion-of-assembled-plasmid.pdf)

## References

1. Fattma A. Ali, Khudhair Al-Daoody AA, Muna M. Najeeb, Media A. Othman, Maryam N. Philip, (2025), The Influence of Restriction Endonucleases for Cloning, *Journal of Clinical Surgery and Research*, 6(1).
   <https://doi.org/10.31579/2768-2757/156>

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# Results

![Barre](https://static.igem.wiki/teams/5653/education/barre.webp)

Interactive Plasmid Map

The Totally [Fe]rocious project aims to develop an alternative to antibiotics in order to deal with antibiotic-resistant *Klebsiella pneumoniae* strains. We propose a Trojan horse approach using aerobactin, a siderophore used by pathogenic *K. pneumoniae* [1], coupled to a gold nanoparticle. The conjugate, once excited with a laser, should be able to induce the death of cells [2]. In the lab, our goal was to express the synthesis pathway of aerobactin in *Escherichia coli*. This pathway, a nonribosomal peptide synthetase-independent siderophore (NIS) synthesis pathway [3], is composed of four enzymes IucA-D [4]. We ordered the sequence for all four genes in the form of gBlocks from IDT. The gBlocks were assembled in the vector by Gibson assembly and transformed in *E. coli*. The siderophore would then be produced and purified using a chromatography-based method and the final product would be quantified by a colorimetric method. The purity would be assessed by mass spectroscopy. Once we have obtained aerobactin in a sufficient amount and at an acceptable level of purity, we would proceed with the coupling of the gold nanoparticles to the siderophore. Ultimately, we would test the conjugate between the siderophore and the nanoparticle to see if, once excited with a laser, it can cause cell death of pathogenic *K. pneumoniae* strains.

## Dry-lab – GEM

---

Our **[modelling](https://2025.igem.wiki/ulaval/model)** and experimental results together provide a comprehensive understanding of how environmental and genetic factors influence citrate and siderophore production in *Escherichia coli*. Using genome-scale metabolic models and constraint-based simulations, we observed clear trade-offs between growth and metabolite secretion. While the wild-type strain achieved high growth rates under neutral conditions, enhanced citrate export occurred primarily in acidic and phosphate-rich environments, and siderophore production remained remarkably stable across most tested conditions. These findings reveal how specific nutrient and pH conditions can be exploited to guide metabolite yields without necessarily improving biomass formation[5].

These results have important implications for our project goals. They confirm that citrate and siderophore pathways can be selectively optimized depending on the environmental context, and that siderophore biosynthesis is inherently resilient—a desirable feature for targeted delivery strategies such as our siderophore–gold nanoparticle conjugate. Moreover, the *ICDHyr* knockout simulations revealed that siderophore synthesis can persist even in the absence of growth, opening possibilities for controlled, non-proliferative production systems.

Not everything went as expected. Some simulation runs initially produced unrealistic flux distributions due to boundary condition errors or missing reaction constraints, forcing us to review model definitions and verify reaction reversibility. Through this process, we learned the importance of model validation, parameter sensitivity testing, and cross-checking with experimental data. These challenges strengthened our understanding of metabolic modelling and improved the reliability of our results.

Overall, these results emphasize the value of combining computational predictions with experimental insight. They also highlight the necessity of transparency, reproducibility, and scientific honesty in modelling—acknowledging uncertainty is as important as presenting strong results. Our work not only supports the feasibility of siderophore-based delivery strategies but also lays the foundation for more predictive and sustainable approaches to metabolic engineering.

## Wet-lab - gBlock Amplification

---

Our first step was to obtain a sufficient concentration of each gBlock to proceed with the Gibson assembly into the extracted and digested pBluescript SK- plasmid. We performed standard PCR protocols following the DNA fragment manufacturer’s recommendations. The results of these PCR amplifications are shown in Figure 1.

The results were satisfactory enough for our needs in the cases of gBlocks 1 and 2. They were both obtained in a sufficient amount to proceed with the Gibson assembly. They were purified on magnetic beads to rid the mixture of the PCR reaction components and the non-specific products. These two were then ready for the Gibson assembly. For gBlock 3, there was too much non-specific amplification and not enough of our goal fragment (around 2,000 bp), meaning that we would not be able to get enough product after purification.

![Citrate pathway](https://static.igem.wiki/teams/5653/results/resultsfig1.webp)

**Figure 1:** Gel electrophoresis of our amplified gBlock fragments as well as Invitrogen’s 1kb+ ladder and High DNA mass ladder. Gel stained with EtBr and picture taken under UV.

We tweaked the PCR protocol for gBlock 3 trying Touchdown (TD) and Inverse-Touchdown (iTD) methods as well as removing the PCR Enhancer from the master mix (see Figure 2). Touchdown PCR consists in hybridizing the primers with the template at a higher temperature during the first cycles and gradually lowering it to Tm - 5°C. This technically leads to more specific amplification in the first few cycles. Inverse-Touchdown PCR, which is the opposite of TD, begins the PCR cycles at a lower hybridizing temperature to make sure the primers hybridize to the template and rising to Tm-5°C. This leads to less specific but easier primer hybridization. The aim of this method was to make sure to obtain an amplicon after the first few cycles. In the end, we found that the iTD protocol without enhancer yielded the best results, so we did multiple reactions with that protocol and pooled the results together after a gel-based purification (we still had too much non-specific amplification to do a column purification).

![Citrate pathway](https://static.igem.wiki/teams/5653/results/resultsfig2.webp)

**Figure 2:** Gel electrophoresis of our different gBlock 3 protocols as well as Invitrogen’s 1kb+ ladder and High DNA mass ladder. Gel stained with EtBr and picture taken under UV.

## Wet-lab - Gibson Assembly & Transformation

---

With all gBlocks in sufficient concentration, we proceeded with the Gibson assembly into pBluescript SK-, followed by a chemical transformation into competent NEB5α *E. coli* cells. The transformed cells were plated on XGAL medium for white/blue screening. A few different positive mutants were selected and they were incubated overnight. We then isolated their plasmid and digested each of them using BamHI and EcoRI. When we did the gel electrophoresis to verify the results, the fragments did not correspond with the ones we were expecting (Figure 3). However, we had multiple repeated digestion patterns indicating that we were able to obtain a certain product in a repeatable fashion.

In our interpretation, this means that during the Gibson assembly, a part of our fragments was successfully integrated to the plasmid in a repeatable fashion. However, somewhere in our gBlock sequences was an element that interfered with the designed assembly and led to an unwanted final product that does not contain the full sequence of all three gBlocks. As we also had trouble with the amplification of gBlock 3, we suspect that that fragment is our problem child. To get some insight into what is happening, we decided to send one of our repeated digestions to Flow Genomics for DNA sequencing. The plasmid from clone 4 was chosen for sequencing.

![Citrate pathway](https://static.igem.wiki/teams/5653/results/resultsfig3.webp)

**Figure 3:** Gel electrophoresis of our different clone digestions with EcoRI (left), BamHI (middle) and no enzyme (right) as well as Invitrogen’s 1kb+ ladder. Gel stained with EtBr and picture taken under UV.

We suspected that it might be ambitious to attempt a Gibson assembly of three 2kb gBlocks at once on an unestablished protocol on limited time, so we also attempted a different assembly method, opting for a PCR Stitch. However, we only had time for one attempt with this method and the stitch of gBlocks 1 and 2 yielded very little product, and not at our desired length of 4 kb (see Figure 4). The protocol we followed for this stitch is the exact same as the protocol for the amplification of gBlocks 1 and 2, except we used the forward primer of gBlock 1 and the reverse primer of gBlock 2.

![Citrate pathway](https://static.igem.wiki/teams/5653/results/resultsfig4.webp)

**Figure 4:** Gel electrophoresis of our PCR Stitch of gBLocks 1 and 2 as well as Invitrogen’s 1kb+ ladder. Gel stained with EtBr and picture taken under UV.

## Wet-lab - DNA sequencing

---

Once the sequencing results came in, we were not shocked to see that the sequenced plasmid was not the expected construction containing all three gBlocks. However, the sequencing did give us some insight on what could have gone wrong. The plasmid extracted from clone 4 comprised 5514bp whereas the expected construction was around 8750bp long. Upon closer inspection, we realized that the sequenced plasmid contained the beginning of gBlock 2 and the beginning of gBlock 3 (see interactive map below). The gBlock 1 and the beginning of gBlock 2 were inserted successfully, but something went wrong and the beginning of gBlock 3 was inserted in the middle of gBlock 2. Since the PCR products used in the Gibson Assembly were all of the right size, the assembly itself was probably not the problem. What could have happened is a recombination event after the transformation of the assembly. The presence of repeated sequences long enough to induce homologous recombination could explain the loss of gBlock 2 and gBlock 3’s sequences.

## Future Outlook

---

These results represent what we were able to obtain in the Wet-Lab for this year’s iGEM cycle. The team’s objective was to tackle the antibiotic-resistant *Klebsiella pneumoniae* strain using a Trojan horse approach. To further develop the analogy, aerobactin was our wooden horse and gold nanoparticles the soldiers hidden inside it. Our unsuccessful attempts at building an expression vector for the enzymes responsible for aerobactin’s synthesis halted us in our steps. The expression of the enzymes and purification of the siderophore represented the first steps in the Totally [Fe]rocious project. Although we did not manage to meet all our objectives, we managed to gain insight on what went wrong and what can be changed in order to go forward with the next steps for this project. As we mentioned earlier, maintaining the expression vector in *E. coli* is most likely where we tripped. Because we were not able to express the enzymes to produce the siderophore, we could not try the purification protocol nor the nanoparticle coupling method. We remain hopeful that we will eventually manage to produce aerobactin and that we will get to try to couple the nanoparticles to the siderophore. Ultimately, the goal would be to test to see whether or not the conjugate between the siderophore and the nanoparticle can act as an alternative to antibiotics in the fight against multiresistant *K. pneumoniae*.

In terms of our next steps, the first thing we will have to solve is the expression system. The Gibson assembly method did not allow us to build a plasmid containing the genes coding for the enzymes required for in vivo synthesis of aerobactin. Since the assembly of all three gBlocks in the vector at once was complicated, we think that assembling them two by two is a more promising way to deal with our issue than redesigning a Gibson assembly from scratch. This is why a PCR stitching method seems like a viable option to us. We tried this method with our current set of primers but it did not work. The use of this method would require us to redesign primers for this specific use. We would assemble the gBlocks two by two, eventually leading to a final assembly step where we would have all three gBlocks lined up in a single fragment and the backbone. This final assembly step can be done by Gibson assembly. The assembly of two fragments instead of four increases our odds of success. Another possible avenue would be to assemble the gBlocks in the backbone by utilizing the recombination ability of *Saccharomyces cerevisiae*. Although this method could work, it would require us to utilize a backbone compatible with the yeast. Once the assembly is done, we would extract the plasmid, amplify the region containing the genes *iucA-D* to clone it in a vector compatible with *E. coli* and transform it into *E. coli*. If our problem really is related to recombination, we would try to transform the assembly in a cloning strain that has limited recombination activity, such as Stbl from Thermo Fischer or NEB Stable from New England Biolab [6,7].

## Interactive Plasmid Map - pBluescript iGEM

---

**Size:** 5,514 bp

**Feature Information:**

Click on a feature to view details

Promoter

CDS/Gene

RBS/Primer

Operator

Origin

Resistance

Misc

If you are interested, here is the complete plasmidic sequence from Flow Genomics:

Plasmid Sequence Box

▶

Plasmid Sequence

## Molecular dynamics

---

Once the various trajectories have been computed, the first step is to assess their stability and convergence towards an equilibrium. Two complementary approaches can be used to evaluate the state of the system. The first focuses on global system properties, monitoring the energy (Fig 6), temperature (Fig 7) and volume (Fig 8). Both the total energy and volume exhibit convergence towards an equilibrium while the temperature remains constant throughout the trajectory. The observed differences in total energy can be largely, if not entirely, attributed to the plug domain (PD), as a greater number of residues naturally contributes to higher structural and interaction energy. This is reflected in a lower potential energy, indicating a more favorable energetic state.

![Citrate pathway](https://static.igem.wiki/teams/5653/model/etotale-croppednter-nonter.webp)

**Figure 6:** Evolution of the total energy of the system during the trajectory. The darker central line represents the smoothed trend of the data, while the lighter background points correspond to the raw computed values.

![Citrate pathway](https://static.igem.wiki/teams/5653/model/temperature-croppednter-nonter.webp)

**Figure 7:** Evolution of the temperature of the system during the trajectory. The darker central line represents the smoothed trend of the data, while the lighter background points correspond to the raw computed values.

![Citrate pathway](https://static.igem.wiki/teams/5653/model/volume-croppednter-nonter.webp)

**Figure 8:** Evolution of the total volume throughout the trajectory. The darker central line represents the smoothed trend of the data, while the lighter background points correspond to the raw computed values.

The second approach relates to the root mean square deviation (RMSD), a metric that quantifies the average distance between atoms of two superimposed structures, effectively measuring conformational differences within the same protein over time [8]. By focusing exclusively on the TBDT, we can compute the various conformational states observed by the protein. A persistent positive RMSD value is expected due to the inherent flexibility of residues with fewer structural constraints. In this case, all trajectories of the systems containing the plug domain converge towards a RMSD of 2 Å, whereas the trajectory lacking the same domain stabilizes around 1.5 Å. The latter thus remains slightly closer to initial conformation, although both display minimal structural dviation from the predicted model. This behavior is consistent with convergence towards a stable state. Only the first replicate of the system with a plug domain shows an early spike in RMSD, which nevertheless remains below 3 Å.

![Citrate pathway](https://static.igem.wiki/teams/5653/model/rmsd.webp)

**Figure 9:** Root mean square deviation (RMSD), of all six trajectories, grouped according to their initial system (with or without the plug domain). All trajectories converge toward a low and stable RMSD value, indicating overall structural stability of the complex. The darker central line represents the smoothed trend of the data, while the lighter background points correspond to the raw computed values.

While the available metrics suggest that the protein complex is stable, the duration of each trajectories, with a minimum of 350 ns, may not be sufficient to fully capture the system in its most stable conformation. Indeed, longer computation, on the order of a 1 μs, would likely provide enough data to more confidently assert that an equilibrium has been reached. By focusing subsequent analysis on the second half of the trajectory, the first half is effectively considered as the adaptation period during which the protein complex would adjust to the various lipids in the membrane.

### Plug domain: structural implication

---

Once the system’s stability has been confirmed, a segment from each trajectories exhibiting a consistent low and stable RMSD was selected as the region of interest for subsequent analysis. From these segments, the corresponding average structures were computed to evaluate the root mean square fluctuation (RMSF), a metric similar to RMSD assessing the fluctuation of each residue throughout a trajectory relative to a mean structure [9]. This analysis identifies regions of the protein complex exhibiting higher mobility. In our case, it provides insight into how the PD influences the overall structure stability.
Figure 10 presents the RMSF profiles for both systems, along with their associated standard errors. The removal of the PD leads to increased fluctuations within the β-strands of the β-barrel, reflected by higher RMSF values. Additionally, the cytoplasmic regions, particularly the β-turns, exhibit higher RMSF values. Altogether, these results illustrate the crucial role of the PD in stabilizing the overall structure, with a particularly strong influence on the cytoplasmic regions.

![Citrate pathway](https://static.igem.wiki/teams/5653/model/rmsf.webp)

**Figure 10:** Root mean square fluctuation (RMSF) of the TBDT for systems with (green) and without (purple) the plug domain. The structural annotations corresponding to the α-helices (αX), the β-strands of the β-barrel (βXX) and other β-strands (SX), are also shown. The top row shows domain and structural annotations from the uniprot entry Q6U607, including the Signal Peptide (SP), TonB Box (TBB), Plug domain (PD), Ligand binding site (LBS), TonB Dependant Receptor β-barrel (TBDR β-barrel) and TonB C-terminal Box (TBCB). The second row shows predicted residue positions in the membrane based on PPM [10], the same method used by CHARMM-GUI [11], with green, orange, and blue indicating cytoplasmic, membrane, and extracellular regions, respectively. Overall, removal of the PD increases fluctuations of the various β-strand of the β-barrel, highlighting PD’s role in structural stability.

### Comparison of Predicted and Empirical Siderophore-TBDT Interactions

---

The interaction between the siderophore-Fe3+ complex and the TBDT represent the first step in the uptake of both the iron ions and the siderophore. The initial analysis involves docking the siderophore complex to the TBDT to assess the ligand’s affinity for binding. Figure 11 compares our predicted interaction (second from the top-left) with experimentally determined TBDT-siderophore complexes. Among the structures shown, only 6Q5E represents an interaction between an NRPS-type siderophore and its TBDT, whereas all others correspond to NIS-type siderophores bound to their respective TBDT. Our predicted complex aligns well with the experimentally observed interactions of other NIS siderophores, with the ligand deeply embedded at the base of the ligand binding site (LBS), while the NRPS interaction (6Q5E) appears to occur near the surface of the TBDT structure.

![Citrate pathway](https://static.igem.wiki/teams/5653/model/siderophores-tbdt-interaction-comparisson.webp)

**Figure 11:** Predicted and experimentally determined interactions between a siderophore and its corresponding TBDT domain. The predicted complex is shown second from the top-left. The remaining structures, arranged from top-left to bottom-right, are taken from the PDB (6I97, 1BY5, 6Q5E, 2W6T, 3QLB et 6E4V).

A closer examination of the predicted interaction between the siderophore by PyMOL [12] and the TBDT is shown in Figure 12. Among the identified contacts, residue Q343 is the only interaction site originating from the β-barrel and located near to the starting residues annotated for the LBS. The other predicted interactions involve residues from the extracellular loop regions (R403, R404 and S707) while residues Y90, R112 and R116 belong to the β2-β3 loop, the polypeptide segment connecting strands β2 and β3. Both of these strands are part of the central β-sheet of the plug domain. Owing to their proximity to the siderophore complex, residues R112 , R403, R404 in multiple interactions. These residues, through their spatial positioning within the TBDT, their sequential arrangement, and their intrinsic chemical nature, likely play a key role in the initial stages of the uptake mechanism.

![Interaction](https://static.igem.wiki/teams/5653/model/aerobactin-interaction.webp)

**Figure 12:** Predicted contact interactions between the siderophe-Fe3+ complex and the TBDT. Residues TYR90, ARG112, ARG116, GLN343 , TYR403, TYR404, SER707 are predicted to interact with the backbone of the siderophore.

## References

1. Kumar, A., Chakravorty, S., Yang, T., Russo, T. A., Newton, S. M., & Klebba, P. E. (2024). Siderophore-mediated iron acquisition by Klebsiella pneumoniae. *Journal of bacteriology, 206(5)*, e0002424.
   <https://doi.org/10.1128/jb.00024-24>
2. Ha, S., Kim, J., Seo, H. W., Kim, L., Yi, Y. S., Seo, S. E., Kim, K. H., Kim, S., An, J. E., Kim, G. J., Ko, K. C., Jun, S., Ryu, C. M., & Kwon, O. S. (2025). Siderophore-Functionalized Nanodrug for Treating Antibiotic-Resistant Bacteria. *ACS nano, 19(5)*, 5131–5145.
   <https://doi.org/10.1021/acsnano.4c06501>
3. Xie, B., Wei, X., Wan, C., Zhao, W., Song, R., Xin, S., & Song, K. (2024). Exploring the Biological Pathways of Siderophores and Their Multidisciplinary Applications: A Comprehensive Review. *Molecules, 29(10)*, 2318.
   <https://doi.org/10.3390/molecules29102318>
4. Bailey, D. C., Alexander, E., Rice, M. R., Drake, E. J., Mydy, L. S., Aldrich, C. C., & Gulick, A. M. (2018). Structural and functional delineation of aerobactin biosynthesis in hypervirulent Klebsiella pneumoniae. *The Journal of biological chemistry, 293(20)*, 7841–7852.
   <https://doi.org/10.1074/jbc.RA118.002798>
5. Du, B., Yang, L., Lloyd, C. J., Fang, X., & Palsson, B. O. (2019). Genome-scale model of metabolism and gene expression provides a multi-scale description of acid stress responses in Escherichia coli. PLoS Computational Biology, 15(12), e1007525.
   <https://doi.org/10.1371/journal.pcbi.1007525>
6. Thermo Fisher Scientific. (n.d.). Competent cells for cloning unstable DNA. Retrieved October 6, 2025, from
   <https://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-applications/competent-cells-for-cloning-unstable-dna.html>
7. New England Biolabs. (n.d.). NEB® Stable Competent E. coli (High Efficiency). Retrieved October 6, 2025, from
   <https://www.neb.com/en-us/products/c3040-neb-stable-competent-e-coli-high-efficiency>
8. Cohen, F. E., & Sternberg, M. J. (1980). On the prediction of protein structure: the significance of the root-mean-square deviation. *Journal of molecular biology, 138*(2), 321-333.
   [https://doi.org/10.1016/0022-2836(80)90289-2](https://doi.org/10.1016/0022-2836%2880%2990289-2)
9. Badar, M. S., Shamsi, S., Ahmed, J., & Alam, M. A. (2022). Molecular dynamics simulations: concept, methods, and applications. In Transdisciplinarity (pp. 131-151). *Cham: Springer International Publishing.*
   <https://doi.org/10.1007/978-3-030-94651-7_7>
10. Lomize, A. L., Todd, S. C., & Pogozheva, I. D. (2022). Spatial arrangement of proteins in planar and curved membranes by PPM 3.0. *Protein Science, 31*(1), 209-220.
    <https://doi.org/10.1002/pro.4219>
11. Jo, S., Kim, T., Iyer, V. G., & Im, W. (2008). CHARMM-GUI: a web-based graphical user interface for CHARMM. *Journal of computational chemistry, 29*(11), 1859-1865
    <https://doi.org/10.1002/jcc.20945>
12. Schrodinger, L. L. C. (2015). The PyMOL molecular graphics system. Version, 1, 8.

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