Felix Biotechnology is developing a microfluidics platform for rapid, high-throughputscreening of therapeutic bacteriophages that target disease-causing bacteria. Federal agencies, multiplecompanies, and infectious disease specialists in major academic medical centers across the US are advancingthe use of phages for a broad range of applications including the treatment of multi-drug resistant bacterialinfections and the prevention of food-borne illnesses. While these efforts show great promise, the narrow hostrange of most phages limits the commercial and clinical potential of phages as a generalized tool. Engineeringphage with expanded host ranges may provide a possible solution, but researchers lack the necessaryunderstanding of the genetic factors that determine host range. Collecting data on genetic variation in host rangeis time consuming, expensive, and low throughput. In preliminary studies, Felix demonstrated 1) the ability toreliably combine bacteria and phage in reproducible ratios in single droplets using a co-flow focusing device, 2)the ability to co-culture bacteria and phage in the droplets and observe phage-specific killing of target bacteria,and 3) the ability to optimize the ratio of bacteria to phage to achieve ⥠99.9% killing in susceptible strains. Inthis proof-of-concept Phase I SBIR, Felix proposes to tag phages and bacteria with unique oligonucleotide-basedbarcodes prior to combining them in droplets, sort droplets where phage successfully kills the bacteria, unify therespective barcodes ("epicPCR") by merging droplets where phage kill bacteria with PCR reagents and thenfusing the barcodes identifying the specific phage and specific bacteria that were involved. The droplets wouldthen be lysed and the pool of hybrid barcodes would be sequenced, giving us information on and sequence-unified amplicons for detecting a lytic pairing. Felix will then demonstrate the ability to distinguish correctly pairedphage/bacteria in a 10 x 10 matrix of different phages and bacteria. Aim 1. Validate the use of oligonucleotidebarcodes for identifying phage/bacteria pairing in droplets. Milestone / Success Metric: Validation of 20 uniqueoligonucleotide-based barcodes (10 phage, 10 bacteria). Aim 2. Demonstrate the ability of barcodes to correctlyidentify phage/bacteria pairs when starting with a matrix of 10 different phages and 10 different bacteria.Milestone / Success Metric: ⥠80% agreement between traditional plaquing assay and the microfluidics assay.Go/No-Go Criterion for Advancing to Phase II: At least 80% agreement between plaquing and microfluidicsassays for identifying phage/host pairs is sufficient to warrant further optimization. Impact-Successful proof-of-concept would support further development of a microfluidics device with a target product profile capable ofscreening a matrix of 1,000 x 1,000 with ⥠95% agreement with traditional plaquing assays. This would provideorders of magnitude more data than current methods, providing the volume of data needed to accurately identifythe genetic basis of host range and engineer phages with expanded host range. These advances couldaccelerate the use of phages as a sustainable first-line treatment for bacterial disease.
Public Health Relevance Statement: PROJECT NARRATIVE
Bacteriophages, natural predators of bacteria, have enormous potential as a tool for treating bacterial
infections, but most bacteriophages only kill one or a few strains of bacteria. To improve the use of these
predators in actual treatments, researchers need to alter the genetic code of bacteriophages to enable them to
kill a broader range of bacteria. This project is designed to develop new tools for rapidly acquiring the large
volume of information researchers need about bacteriophage / bacteria interactions to determine which changes
they can make to improve the array of bacteria each type of bacteriophage can attack.
Project Terms: |
