Introduction

With this collection, we would like to share the protocols used by our team for our project. By making these protocols available, we hope to contribute to the iGEM community and support others who work with similar techniques. The protocols are open for everyone to use and are written in a user-friendly way to ensure accessibility and ease of application.

In our project NRPieceS, we are using molecular machines called Non-ribosomal Peptide Synthetases (NRPS) to design new peptide antibiotics. NRPS are huge modular enzymes that produce peptides. Each module is responsible for selecting and adding a specific amino acid to the growing peptide chain^[1]. By exchanging these modules, we can design entirely new peptides with potential antimicrobial activity. To make this approach widely accessible, we are building a toolbox of interchangeable modules.

The first steps of our project focused on making NRPS accessible to build our peptide library. To achieve this, we relied on cloning techniques, which are detailed in Section Cloning. We then applied the techniques described in Section Expression to evaluate the expression of our newly designed peptides. Next, we turned to bioactivity testing against ESKAPE microorganisms — the most critical pathogens in the fight against antimicrobial resistance. These protocols are provided in Section Bioactivity Testing. To further enhance the effectiveness of our peptides, we also developed a drug delivery strategy to improve targeting. The relevant protocols are presented in Section Drug Delivery. Section Measurements describes the LC-MS and HPLC protocols in detail, which are essential for the identification and purification of our compounds, respectively. Last but not least, all media, buffers, and solutions required for our experiments are compiled in Section Buffers, Media and Additives.

Be safe! Always prioritize safety when working in the laboratory. Wear a lab coat and protective gloves, handle chemicals with care, and follow all established safety guidelines. Most of the protocols described below were carried out in a BSL-1 laboratory, while the bioactivity testing was conducted in a BSL-2 laboratory. Ensure that you are familiar with and comply with the specific safety instructions for each laboratory environment.

Cloning

Introduction

Our project focused on making non-ribosomal peptide synthetases (NRPS) more accessible and on building a diverse peptide library. To achieve this, we employed a combination of cloning techniques. The NRPieceS library was constructed from native NRPS clusters, which were first transformed into acceptor vectors. In parallel, we designed donor vectors containing interchangeable modules. The construction of both acceptor and donor vectors was carried out using the Gibson Assembly method (Fig. 1). By subsequently combining these vectors through Golden Gate cloning, we successfully established the NRPieceS library. Both cloning techniques, Gibson Assembly and Golden Gate Assembly, are described in more detail in the Cloning protocols.

Schematic Workflow

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Expression

Introduction

After designing and constructing our NRPieceS library using cloning methods, we aimed to test the expression of our newly designed peptides (Fig. 2). E. coli was chosen as the host organism due to its simplicity, rapid growth, and high genetic flexibility, making it an ideal production platform^[2]. Both the previously known peptides from native clusters and our newly designed variants were produced in E. coli. Depending on the requirements of each experiment, production was carried out at different scales, including small-scale production for bioactivity testing, high-throughput screening for the entire NRPieceS library, and large-scale production.

Schematic Workflow

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Bioactivity Testing

Introduction

As the overall goal of the NRPieceS project is the discovery of novel antibiotics, testing our peptides against the relevant organisms is an integral aspect. These pathogens are often referred to as ESKAPE organisms, which is an acronym for the bacterial strains that are known for evolving clinically relevant resistances against antibiotic treatment (Fig. 3).

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We tested our peptides against representatives of the ESKAPE organisms or close relatives of such and a high-sensitive Bacillus subtilis strain which is used in diagnostics for the detection of even minor antibiotic concentrations. The strains were plated on LB and exposed to the peptides in either a drop spot or disc assay. After an overnight incubation, it was examined if the peptides caused bacterial growth impairment by measuring the inhibitory zones in comparison to the controls (Fig. 4).

Schematic Workflow

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Drug Delivery

Introduction

Gram-negative bacteria are notoriously hard to kill because their outer membrane blocks many antibiotics. A way to bypass this barrier is the Trojan Horse strategy, where antibiotics are coupled to molecules that bacteria actively import, such as siderophores or sugars. This approach allows compounds to slip past defenses and reach their targets^[3]. To improve targeting, we attempted to conjugate our peptides to a siderophore via click chemistry. This method requires building blocks with reactive groups such as terminal alkynes^[4]. . For this purpose, we synthesized an amide containing both catechol and alkyne functionalities by coupling protocatechuic acid with propargyl amine. Within our NRPieceS project, several conjugation strategies were tested, including direct siderophore and azide handle feeding, as well as incorporation of 4-azido-L-phenylalanine (AzF) by adenylation domains. After multiple attempts, we successfully introduced the azide handle in our peptide and carried out click chemistry with it (Fig. 5).

Schematic Workflow

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Measurements

Introduction

Reliable and reproducible measurement is a keystone of synthetic biology and enabling quantitative comparison across experiments and laboratories. In our NRPieceS project, we implemented LC–MS with rigorous calibration curves, blanks, and controls, and complemented it with HPLC purification and NMR analysis to confirm compound identity and structural features. LC–MS served as the quantitative backbone of our workflow, while HPLC and NMR provided complementary support for purity and structural confirmation, creating a robust framework for reliable measurement of NRPS-derived products (Fig. 6).

Schematic Workflow

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Accessible Workflow for Peptide Discovery

Introduction

We recognize that not every lab has immediate access to advanced analytical instruments such as LC-MS, HPLC, or NMR. To make our approach more accessible, we developed a tiered workflow designed to lower the entry barrier for NRPS screening. This workflow encourages teams to begin with straightforward functional assays, such as bioactivity tests to quickly identify promising candidates, without relying on specialized equipment. Only when a candidate shows measurable activity do we recommend moving on to more resource-intensive chemical analyses like LC-MS or NMR. By structuring the process in this way, even teams with limited infrastructure can meaningfully participate in peptide discovery and focus their efforts and resources where they matter most. In the following section of this collection, we walk you through our full production workflow — all the way from culturing to bioactivity testing. You can follow these steps as a practical guide to produce and test your own peptide extracts. This approach helps you quickly spot promising antimicrobial candidates before deciding whether to invest time and resources in advanced techniques like LC-MS or NMR.