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Team:IIT Roorkee/Description




In the coming few decades, when bacterial infections would become untamed due to drug resistance , we are likely to witness a ten-fold increase in death rates and indefinite damage to the global economy in comparison to the pandemonium caused by the current coronavirus pandemic. Hospital acquired infections which are antibiotic resistant are the leading cause of most of the ICU (intensive care unit) deaths, and therefore there is an imperative need to build alternative solutions to treat bacterial infections and and reduce the dependence of healthcare systems on antibiotics. Our project provides a narrow-spectrum treatment for the multidrug resistant pathogen, Acinetobacter baumannii, by the engineering and combination of natural antibacterial molecules-bacteriophages and pyocins. Pyocins are bacteriocins produced by P. aeruginosa. Our key engineering work is in the design of novel antimicrobial protein complexes called Seekercins, made by fusion of the pyocins and bacteriophage tail fiber proteins. We have designed a web-based software tool, called TailScout, for conveniently designing these fusion proteins. Further, a genetic construct was designed for production using standard synthetic biology tools. The chassis we have chosen is E. coli. For validation of our system, we are targeting the pathogen Acinetobacter baumannii. Through our project Pyomancer, we are also propagating the message that the time has come for a paradigm shift to narrow-spectrum antimicrobial therapies in the healthcare sector so as to regulate the damage done by the continuous use of broad-spectrum antibiotics.


The COVID-19 pandemic led to a massive disruption in the global public health system. This helped us closely to understand the kind of threat we are headed towards as antibiotics continue to fail and no new treatment strategies are available. We found this reality scary and deeply concerning. On investigating the issue at the ground level, through doctors and ICU specialists, we realised that the possibility of slowing down the development of resistant bacterial species is far from feasible. Blind prescription of broad spectrum antibiotics and its consequences nudged us to tackle this issue from a treatment point of view and use our engineering skills and understanding of biology to develop novel protein complexes which are unconventional from how the antimicrobials currently work. Our key inspiration was the TAU Israel's iGEM 2019 project, Pyo-Pyo, that illustrated that the relatively short evolutionary distance between pyocins and certain bacteriophages enables us to use phage tail fibers to choose a target bacterium. We built upon the concept introduced by TAU Israel's iGEM Team and explored new realms unique to our project.

We acknowledged that the heightened sensitisation of people around the world about pathogens and hygiene provided a good opportunity to propagate WHO’s Global Action Plan against AMR. As a result, we could deliver the pertinent message through our human practices and public engagement activities and received good feedback from the community. Further, the challenges posed by the COVID-19 (closed labs and remote work), encouraged us to explore protein modelling, invest in the development of a software and try our hands on Machine Learning.


R-type pyocins are bacteriocins which are evolutionarily related to the Myoviridae phage tails and are produced by Pseudomonas aeruginosa. Bacteriocins are high molecular weight bactericidal protein complexes and differ from bacteriophages mainly because they lack genetic material . Five different R-type pyocins have been identified, and each type has a specific killing spectrum. Under stressful conditions, P. aeruginosa produce pyocins which are released by cell lysis (due to the presence of lysis cassette present in the gene cluster) and these inhibit the growth of competing bacteria.

R-type pyocins consist of a tube-like core, surrounded by a sheath. At one end of the core, there is a complex baseplate structure, and attached to this baseplate are the tail fibers that can specifically bind to a receptor on the target bacteria's membrane. Pyocins attach to receptors on the bacterial cell wall and penetrate it with a hollow tube, leading to depolarization, i.e. dissipation of membrane potential and subsequent cell death. Hence, their killing mechanism is similar to the DNA injection mechanism of Myovirus phages. They have a high killing efficiency but a very narrow host spectrum.

A. baumannii

A. baumannii is an opportunistic gram-negative bacterium and responsible for a broad range of nosocomial infections, the most important of which are ventilator-associated pneumonia and bloodstream infections, and the mortality rates can reach 35%, upto 84% in XDR cases. Currently, A. baumannii has developed resistance to almost all known antibiotics. Acinetobacter baumannii is a particularly challenging pathogen because it is associated with a high degree of resistance, and it is difficult to eliminate its environmental reservoir in healthcare settings with conventional measures. The development of drug resistance in A. baumannii is fast. It took only 11 years for the percentage of resistance to imipenem to increase from 23.8 to 73.9% . Thus, it is expected that resistance to tigecycline and colistin could be common in another ten to 20 years time if no preventative measures were taken, which may result in an absolute “post-antibiotic era” for pan-antibiotic resistant A. baumannii infections. There is a continued inadequacy of monotherapy for resistant A. baumannii infections due to a lack of highly effective drugs and/or the lack of adequate site concentrations of drugs. It is evident that to deal with such bacterial infections, traditional antibiotics do not add much value and further exacerbate the damage. Our solution addresses this gap in the treatment of these infections and establishes a treatment strategy for handling the pathogen.


A New Class of Antimicrobial Complexes

Our solution is the development of ‘Seekercins’, novel antibacterial protein complexes by engineering of R-type pyocins to target specific drug resistant bacteria. It involves the rational design of fusion tail fibers, made from genes of wild-type pyocin tail fibers and tail fibers of phages. The binding of a phage to its receptor is mediated by the binding domain of their tail fibers. We aim to exploit the structural similarity between pyocins and phages to effectively retarget pyocins. In our strategy we developed a synthetic R-pyocin involving a rationally designed chimeric R-pyocin tail fiber with the tip modified to bind to A. baumannii. To achieve this, we replaced the lectin-fold head region of R-pyocin tail fiber with a similar lectin-fold binding domain from A. baumannii specific bacteriophage AP22 tail fiber. The chimeric R-pyocin tail fiber, along with other genes in the synthesis cluster, will be cloned into an E. coli expression vector under inducible promoter. The AP-22 tail fibers of Bacteriophage will allow specific attachment to the A. baumannii's surface proteins called Lipooligosaccharides. To achieve this, we made a fusion tail fiber by fusing the N-terminal portion of the pyocin tail fiber to the C-terminal part of the tail fiber of phages that naturally target these bacteria.. This method makes it possible to create pyocins that specifically target and kill the target bacteria with high efficiency, provided the appropriate phages are found. Further, we plan to purify the synthetic pyocins from the recombinant E. coli and utilize them to kill A. baumannii in MDR infections.

To read further on the protein design process and our plasmid assembly refer here

Furthermore, we provide a modular, standardized system for the production of such protein complexes that could be designed for any possible bacterial species, including MDR by delivering:

  • A standard expression plasmid for the convenient production of such fusion proteins
  • A supporting software program called TailScout, containing cross-referenced libraries of hosts and their phages that will allow us to find the required tail fiber sequences easily and design fusion tail fibers

Our engineered E. coli systems are protein producing machineries, which will be used to mass produce fusion proteins. On purification they can be added into formulations that will be administered at places where antibacterial treatment is required. This can range from food contamination, crop diseases to MDR infections in humans. It will require a considerable amount of research and experiments, to conclude if the drugs designed this way could be administered as therapeutics.

Our projects aims to provide new parts to the Registry that would allow for productions of pyocins by any future iGEM team or anyone in the synthetic biology community.



Pyocins have mainly remained unexplored as antibacterial treatments. They employ single-hit bactericidal kinetics (one pyocin molecule is theoretically capable of killing one bacterium) which make them extraordinarily potent antimicrobials.


These proteins can be rapidly designed to target virtually any gram-negative bacterium.

Targeted specificity

It is a targeted treatment that will kill only selective bacteria and not cause any damage to off-target species. This treatment will cause minimal unintended collateral damage to the rest of the microbiota.

Mass Production

As pyocins are a protein product, they can be mass-produced and stocked like related protein-based biotherapeutics.

Slow Resistance Development

Resistance to pyocins is less studied and might be less frequent (avoiding antibiotic resistance mechanisms, phage resistance, and even horizontal gene transfer), which can prove to be an excellent advantage over antibiotics and phages. Our engineered protein targets surface accessible virulence or fitness factors like LPS. Even if some rare bacteria develop resistance to the targeted protein by altering the virulence proteins, it would mean that the virulence factors are compromised, and they may not be pathogenic anymore.

Scope of implementation

  1. Food Contamination
    The most significant contributor to the rapid evolution of AMR species and superbugs is the poultry industry, where high amounts of antibiotics are used in an uncontrolled manner for treating the animals. Bacterial contamination of food products is a growing concern, especially for poultry-based products, because of the heavy use of antibiotics. Once our proteins are validated, they can be administered to control bacterial contamination of dairy and poultry products, significantly reducing the chances of transfer of infection.

  2. Crop Protection
    During our literature review, we came across a severe apple disease called Fire Blight caused by Erwinia amylovora (Enterobacteriaceae) . This disease is highly infectious, destructive, and damages complete orchards within a few days, causing a tremendous economic loss. Our highly specific proteins can be designed to kill Enterobacteriaceae by finding a lytic phage and combining it with naturally produced pyocins.

  3. Therapeutic drug for MDR infections
    In an absolute “post-antibiotic era,” we need an alternative treatment strategy, and our solution has a considerable potential that can be harnessed with an appropriate plan of action for the increased tests and trials. Once we are able to produce results and data in-vitro, we can run more experiments to characterize the protein production, increase its expression levels, understand in-vivo responses, and ultimately escalate to clinical trials. We envision it as a revolution in the pharmaceutical industry.

  4. Microbiome Editing Tool
    The current unavailability of tools to selectively target bacteria without affecting the diversity poses a limit to how much we can validate the proposed relationship and interaction effects of a bacterial community in environment, health and disease. Our product is unique in the sense that it can be designed to be species-specific or even strain-specific, as per the choice of the designer. Thus, we can potentially elevate this strategy for use in scientific studies and research on microbiomes to selectively kill a bacterium from its environment and study its effects, interactions, and consequences.


  1. https://www.intechopen.com/books/antimicrobial-resistance-a-global-threat/antibiotic-use-in-poultry-production-and-its-effects-on-bacterial-resistance
  2. https://extension.uga.edu/publications/detail.html?number=C871&title=Fireblight:%20Symptoms,%20Causes,%20and%20Treatment
  3. https://www.annualreviews.org/doi/abs/10.1146/annurev-virology-101416-041632
  4. https://www.mdpi.com/1999-4915/10/8/427’
  5. https://www.cdc.gov/hai/organisms/acinetobacter.html
  6. https://academic.oup.com/cid/article/42/5/692/2052763
  7. https://2019.igem.org/Team:TAU_Israel
  8. https://www.researchgate.net/publication/5411516_Retargeting_R-Type_Pyocins_To_Generate_Novel_Bactericidal_Protein_Complexes