Week 1 HW: Final Project Ideas

(1) What you would like to biomanufacture and why? An often-quoted estimate predicts that antibiotic resistant infections will outpace cancer and heart disease as the global leading cause of death by 2050. Resistant bacteria are especially prevalent in hospital settings, where high antibiotic concentrations and large, diverse populations of pathogenic bacteria combine (within infected patients, in drains, etc) to drive rapid resistance evolution. Resistant bacteria are difficult to eliminate not only because 1), they, by definition, cannot be treated via traditional antibiotics, but also because, 2), they tend to form dense, sticky, and highly resilient biofilms. I want to biomanufacture something that combats antibiotic resistant pathogens – possibly via a phage-therapy based approach, taking advantage of automated and cloud-lab systems to design, engineer, and test a variety of modified phage at a large scale and create/identify promising anti-biofilm candidates against various bacterial pathogens.

(2) What living system would you use? I would likely use Mycobacterium smegmatis as my host bacterium for phage engineering, testing, and large-scale phage production. (And I would use the large repository of Mycobacteriophages available through the HHMI SEA-PHAGES program as a starting point for engineering anti-biofilm phage therapies.) Mycobacterial species can form resilient biofilms that contribute to devastating hospital-acquired infections. M. smegmatis, as a BSL1, non-pathogenic, and relatively fast-growing Mycobacterial species, offers an easier and safer system that could discoveries relevant to treating pathogenic Mycobacteria such as M. tuberculosis and M. abscessus. (I.e., design principles that I discover apply to the anti-biofilm properties of M. smegmatis phages may help us eventually engineer therapies for M. abscessus.)

(3) How would you use automation? I would use an Opentron liquid handling robot to automate the phage engineering and cloning-based parts of this process – so as to make it possible to engineer, reboot, and test many slightly different engineered variations of the same phage. I would likely also use a larger Cloud Lab system for the testing and measurement stages: I would need an automated and standardized way of producing biofilms with qualities of interest, potentially on surfaces designed to mimic a hospital or patient setting, and a way to measure the extent to which the biofilm was cleared by engineered phage – and then to select for and amplify/propagate more of, acquire, and sequence the effective phage.

(4) How much would you need to make, and how long would it take? After engineering a particularly effective anti-biofilm phage via the methods above, I could expand my system to produce the relevant phage at a large scale for treatment. Phage therapy dosage can vary – one review says that they tend to be 10^{9 - 10}10 PFU (plaque forming units) per dosage for acute infections (https://pmc.ncbi.nlm.nih.gov/articles/PMC11870740/#sec10). Acute respiratory infections can require up to 4 dosages per day, so 4 x 10^{10 PFU /day. If the patient must be treated for 7 days, they require 28 x 10}10 PFU total – aka 28 x 10^{10 phage. Let’s say that the typical mycobacteriophage burst size is 150 phage, and each 200 ul well in a 96 well plate holds ~10}7 bacterial cells, and we add inoculate each well with enough phage to kill half of the bacteria – we would end up with ~7.5 x 10^8 phage per well(?). If so, we would need to run at least ~373 infections in 200 ul wells to produce enough phage for one patient.

Timing-wise, each well-infection could run in 1-2 days and then be harvested to infect the next round. If we started with enough pure phage lysate, we could, e.g., run 96 initial parallel infections in a plate per patient in 24 hrs, then harvest the filtrate and start 2-3 new infections per harvested well in order to obtain the remaining phage in another 24 hrs.

(5) What don’t you know yet? What is the best approach to automate engineering of mycobacteriophages? What cloning techniques would be optimal to use? How might I get started with identifying engineering modifications that would provide anti-biofilm properties? What technqiues and growth conditions can I use to grow test biofilms at large scale in a automated Cloud Lab setting?

Testing