Contribution

Our team is the first to recognize and utilize darobactin’s antibiotic activity against Gram-negative pathogens to combat Erwinia amylovora. In addition, our project is the first to associate an inducible promoter with darobactin production, addressing concerns of excessive antibiotic production being released into the environment and mitigating the risk of antibiotic resistance. Our new composite part, detailed below, contributes to the future of  iGEM teams by providing precedent for the use of Dar A-E, a lac promoter system, and a mutated BamA sequence for the production of darobactin.

• Building off of tedious scientific work and plasmid design by the Lewis Lab at Northeastern University (Imai et. al), our proposed plasmid designs adds an infection triggered promoter and mutant BamA sequence to act as a kill switch
• Our composite part BBa_K5145006, combines the darA-E sequence, parts of the lac operon (lac promoter, lac operator, and lac inhibitor), and the mutated BamA sequence (all described in the table below)
• BBa_K5145005, a mutant BamA sequence, is a new part that allows for the synthesis of darobactin in gram-negative bacterial vectors.
• Additionally our composite part BBa_K5145006, includes the basic part BBa_K4846004 encoding for darE added to the parts registry by FSHS GD 2023 iGEM team. Due to editing limitations, we were not able to document our contributions onto BBa_K4846004, however we did end up creating a new part BBa_K5145004, that explains how we used it in our plasmid.
• Our idea is precedent for further research into infection sensitive plasmids in the agricultural industry.

Kim Lewis Lab at Northeastern University 

• In 2019, researchers at Northeastern University’s Lewis Lab discovered, sequenced, and characterized darobactin, an antibiotic that selectively kills Gram-negative pathogens. In their groundbreaking paper titled “A new antibiotic selectively kills Gram-negative pathogens,” they described how darobactin targets essential outer membrane proteins in Gram-negative bacteria, effectively circumventing traditional antibiotic resistance mechanisms. The darobactin plasmid as well as purified darobactin were generously supplied to us by the Lewis Lab, providing a crucial foundation for our project and marking a significant contribution to advancing the fight against antibiotic-resistant bacteria. (Imai et. al)

Guangdong Country Garden School iGEM Team (2023)

• In 2023, the Guangdong Country Garden School iGEM team addressed antibiotic resistance by combining the antimicrobial peptide (AMP) nisin with darobactin. They created a chimeric fusion peptide that exhibited both anti-Gram-positive and anti-Gram-negative activities, expressing it in E. coli and successfully verifying its function. Since both nisin and darobactin are ribosomally synthesized and post-translationally modified peptides (RiPPs), they used similar biosynthetic pathways to co-express them. This team’s work on darobactin laid a foundation for our project and provided important insights into combining AMPs for enhanced antimicrobial action.

Other iGEM teams have previously worked to combat Erwinia Amylovora. We aimed to learn from their efforts while pursuing a novel idea. 

• In 2017, the Tec-Chihuahua team engineered a strain of E. amylovora that produces three enzymes that disrupt quorum sensing, intracellular messaging, and cell mobility. The results of this project showed that E. coli can serve as a suitable model during the design process, but the final design of a biocontrol organism must be fit for its target environment. This project produced an E. amylovora strain with reduced virulence. Upon reasoning that the wild-type E. amylovora may outcompete the non-virulent version, we decided to engineer a different bacteria to combat E. amylovora.
• The 2023 USC team engineered a bacteriophage to attack E. amylovora. This project showed the importance of specificity, so we are using an environmentally-inducible promoter that is triggered when needed. We engineered bacteria rather than bacteriophages so the bacteria could work to outcompete E. amylovora along with administering our treatment.
• The 2013 University of Nevada team produced endolysin proteins to target Erwinia Amylovora. This project demonstrated how special consideration must be taken to design a treatment for gram-negative pathogens. While this project chose to deliver their treatment straight to the bacteria, we decided to engineer a bacteria to continuously produce our treatment as needed over time.

Another iGEM team has used darobactin before in their project. 

• In 2023, the FSHS-GD team genetically combined nisin with darobactin and expressed this engineered peptide in E. coli. This project focused on the use of darobactin as an antibiotic to fight bacterial infections and minimize antibiotic resistance. Their project demonstrated the efficacy of darobactin, and while they chose to merge it with nisin, we are choosing to produce darobactin on its own in a different bacterium, P. agglomerans.