Projects

Final projects:

• Individual Final Project
• Group Final Project

Final Project - Ideas/Drafts

Idea: Space Phage Supreme

Section 1: Abstract

• Phage therapy’s potential to treat novel bacterial infections has generated increased attention in recent years, terrestrially and in space health research. Recent research from University of Wisconsin Madison demonstrated the unique impacts of microgravity on Escherichia coli bacteria and T7 bacteriophage interactions, particularly on the distribution of genetic mutations across the T7 bacteriophage genome ¹. Understanding unique microgravity-derived insights on bacteriophage mutations and bacteriophage bacterial interactions could yield phage therapeutic insights terrestrially and for future space travelers. Accordingly, this research aims to extend the University of Madison, Wisconsin’s research by proactively apply analogous microgravity-derived phage mutations in a new bacteriophage. The working hypothesis of this research is that proactively applying analogous microgravity-derived phage mutations in a new bacteriophage will yield similar increase in bacteriophage fitness and lysing.

To implement this analogous research, the plan is to:

• Perform targeted, direct engineering involving Ribosome Binding Site (RBS) and tip-domain substitutions in Mycobacterium smegmatis (M. smegma) bacteriophage to improve host fitness and lysing via phage recombination or CRISPR-based techniques
• Test mutated M. smegma bacteriophage host range and infection efficiency

The methods for achieving the specific aims referenced above include:

• Single-site mutagenesis of relevant RBS/tip domain proteins
• Deep sequencing to map variant fitness pre/post-selection
• Whole genome sequencing for de novo mutation identification
• Use host plating assays for lysing analysis

Section 2: Project Aims:

• Experimental Aim: The first aim of this project is to demonstrate if/how analogous E. coli microgravity bacteriophage mutations manifest in M. smegma bacteriophage via single-site mutagenesis of relevant RBS/tip domain proteins, followed by deep and whole genome sequencing as necessary. This will help discern insights on M. smegma bacteriophage fitness and lysing

• Developmental Aim: Follow-on experiments showcasing comparable mutations across several additional bacteriophage species, with the aim of discerning which microgravity bacteriophage mutations can instigate positive terrestrial human health outcomes (specifically improved bacteriophage fitness and lysing)

• Visionary Aim: ‘Plug and play’ (ideally bidirectional [terrestrial and space-based]) catalog of microgravity-derived high-fitness bacteriophages for use against nth forms of bacterial infection. This would help confront the challenge of novel (or routine) space-based bacterial infections and lack of corresponding antibiotics

Section 3: Background:

• Briefly summarize two-peer reviewed research citations relevant to your research
  • In ‘Microgravity reshapes bacteriophage–host coevolution aboard the International Space Station’ University of Wiconsin Madison researchers reported on the dynamics between a T7 bacteriophage and E. Coli after microgravity exposure aboard the International Space Station (ISS) ². Their results indicated delayed phage activity, but ultimately the emergence of several novel mutations across the bacteriophage, which when replicated terrestrially, improved lysing. In ‘Impact of simulated microgravity in short-term evolution of an RNA bacteriophage’ researchers from the Centro de Astrobiología and Universidad Autónoma de Madrid also discovered similar delayed phage activity when RNA bacteriophage was exposed to a terrestrial simulated microgravity environment ³. Both studies indicate novel phage activity due to microgravity exposure.
• Explain how your project is novel or innovative
  • My project seeks to extend learnings on bacteriophage microgravity exposure to determine how microgravity-derived phage mutations can proactively apply to improve terrestrial phage fitness (including but not limited to lysing). If successful, this project will help demonstrate the utility of microgravity in terrestrial phage therapy development. If successful, it might also help create a bidirectional virtuous cycle between microgravity-derived insights, terrestrial phage therapy, and non-terrestrial phage therapies for long-duration space missions
• Explain why your project matters and what impact it could have
  • This project attempts to solve the problem of proactively improving phage fitness. Improving phage fitness matters as it’s crucial to making phage therapies a viable alternative to traditional antibiotics, particularly in remote, resource-constrained environments like a long-term space exploration mission. Creating the bidirectional virtuous cycle referred to in the answer to the previous question could advance public health and wellbeing in several ways. It could combat antimicrobial resistant (AMR) bacteria while giving humanity a means of dealing with space-based infections when nth volumes of antibiotics or standard pharmaceuticals may be in short supply or logistically unfeasible to transport. In helping bidirectional virtuous cycle, this research will advance our knowledge of customizing terrestrial bacteriophage for improved fitness based on microgravity-derived insights
• Describe the ethical implications associated with your project and identify relevant ethical principles (i.e., non-maleficence, beneficence, justice, or responsibility)
  • Improving phage fitness might have unintended consequences, as significantly fit phage could lyse bacteria that are important to function of human microbiomes. Therefore, this project intends to follow the principle of non-maleficence. In practice this means our research will be conducted in low-biosafety (BSL) environments on M. smegma and will focus on improving phage fitness to combat AMR bacteria. This research will also uphold the principle of beneficence by making the results of our research publicly available. The measures taken to ensure this project aligns with ethical principles are mentioned in passing in the previous paragraph and elaborated upon here. The research will be conducted in low-BSL settings, and its results will be made publicly available. Any/all researchers associated with this project will comport with all appropriate statutes in maintaining lab safety at all times. While there could be unintended consequences of publicly sharing this research, any/all researchers associated with this project will share what is strictly necessary within the scope of this project’s research aims. Any/all discussion of using bacteriophages to deliberately alter human microbiomes for adverse health outcomes will not occur.

Section 4: Experimental Design, Techniques, Tools, and Technology

• Create a detailed experimental plan for your final project. Include a timeline for each part of your experimental plan (i.e., how long you expect each step in your final project to take)

NOTE: This is Claude-derived per tool in this HTGAA hyperlink ⁴ and tentative

The experimental workflow spans 4–6 weeks and is divided into four phases: (1) construct design and ordering, (2) library generation and recombineering, (3) automated screening, and (4) validation and analysis.

Detailed Workflow Workflow (≥15 Steps)

Step 1 — Bioinformatic identification of D29 tail spike host-range determinant residues

• Method: Structural homology modeling of D29 gp10 (tail spike) and minor tail proteins using AlphaFold2; alignment with T7 tip domain; identification of surface-exposed residues likely involved in glycolipid receptor contact
• Automation: Computational (no lab automation required)
• Expected result: 8–15 target residue positions for combinatorial diversification

Step 2 — Design of combinatorial tail spike variant library

• Method: Design of 96–192 D29 tail spike variants encoding combinations of microgravity-inspired substitutions (hydrophilic/aromatic substitutions at receptor-contact positions, guided by Huss et al. microgravity fitness preferences); design includes full upstream/downstream homology arms (500 bp each) for recombineering
• Automation: Computational design; Twist Bioscience order submission
• Microplate: N/A (ordering stage)
• Expected result: Finalized Twist order of combinatorial tail spike library as linear dsDNA fragments

Step 3 — Twist Bioscience DNA synthesis and delivery

• Method: Order combinatorial library as Twist Oligo Pools (clonal gene synthesis for top 12 priority variants as individual whole-plasmid orders for validation constructs); also order pJV53 recombineering plasmid backbone as a Twist whole-plasmid synthesis
• Automation: External (Twist Bioscience)
• Expected result: Pooled oligo library + 12 individual constructs delivered

Step 4 — M. smegmatis mc²155 electrocompetent cell preparation

• Method: Grow mc²155 to mid-log in 7H9 media, wash 3× with 10% glycerol, aliquot and freeze at −80°C
• Automation: Manual preparation; HiG Centrifuge for pelleting
• Microplate: N/A
• Expected result: Electrocompetent mc²155 cells ready for recombineering

Step 5 — pJV53 recombineering plasmid transformation into mc²155

• Method: Electroporate pJV53 (encodes Che9c gp60/gp61 recombinases under acetamide-inducible promoter) into mc²155; select on hygromycin plates; confirm by colony PCR
• Automation: Manual electroporation; ATC Thermal Cycler for colony PCR confirmation
• Microplate: 96-Armadillo-PCR-AB2396X for colony PCR
• Expected result: mc²155/pJV53 recombineering-competent strain confirmed

Step 6 — Recombineering: introduction of tail spike library into D29 phage genome

• Method: Infect mc²155/pJV53 with wild-type D29 at MOI 0.1 in presence of tail spike library linear DNA (500 bp homology arms); induce recombinases with acetamide at time of infection; harvest phage lysate after 4h lysis
• Automation: Inheco Plate Incubator for controlled 37°C incubation; BioshakeD3000 for mixing during induction
• Microplate: 96-v-eppendorf-951033502-deep (deep well for liquid culture volumes)
• Expected result: Recombinant phage library in lysate; estimated recombination frequency 10⁻⁴–10⁻³

Step 7 — Primary plaque assay screen on mc²155 (permissive host, library QC)

• Method: Serial dilution of phage library lysate; plaque assay on mc²155 lawn in 7H9 top agar; count plaques to confirm library titer; pick 48 random plaques for Sanger sequencing of tail spike region to confirm library diversity
• Automation: Echo525 for serial dilution transfers into 384-well plate; Multiflo for top agar dispensing
• Microplate: 384-flat-corning-3640 for dilution series; omni plates for agar overlays
• Expected result: Library titer ≥10⁸ PFU/mL; sequencing confirms >60% variant recovery

Step 8 — Automated host range screen: plaque assay across 3 M. smeg strains

• Method: Plate library phage against (a) mc²155 (permissive control), (b) M. smeg ATCC 607 (naturally more resistant to D29), (c) M. smeg strain with modified cell wall glycolipids; score plaques at 24h and 48h; calculate relative plating efficiency (EOP) for each variant on each strain
• Automation: Echo525 for phage dilution transfers; Multiflo for bacterial lawn + top agar dispense; Spark Plate Reader for OD-based bacterial growth confirmation pre-plating; Plateloc with A4s breathable seal for incubation; Cytomat for 37°C shaking incubation of liquid cultures
• Microplate: 384-flat-corning-3640 for plaque assay screens; 384 Greiner black-well clear-bottom for OD600 bacterial growth monitoring
• Expected result: Identification of 3–10 variant plaques with EOP >10× WT D29 on resistant strains

Step 9 — Plaque isolation and phage stock preparation of top variants

• Method: Pick top 12 expanded-host-range plaques from Step 8; propagate individual phage stocks on mc²155; titer by plaque assay; extract phage genomic DNA for sequencing
• Automation: Echo525 for phage propagation setup; PHERAstar FSX for high-sensitivity titer confirmation via absorbance
• Microplate: 96-round-axygen-pdw11cs-halfdeep for phage propagation
• Expected result: 12 purified expanded-host-range D29 variant stocks at ≥10⁹ PFU/mL

Step 10 — Adsorption rate assay for top 12 variants

• Method: Mix each variant phage with each target M. smeg strain at MOI 0.01; centrifuge at defined time points (2, 5, 10, 20 min) to pellet bacteria + adsorbed phage; measure unadsorbed phage in supernatant by plaque assay; calculate adsorption rate constant k
• Automation: Echo525 for phage + bacteria mixing in 384-well format; HiG Centrifuge for pelleting; Spark Plate Reader for OD600 monitoring of bacterial density
• Microplate: 384-flat-perkin-elmer-60082XX-shallow for adsorption kinetics timepoints
• Expected result: Top variants show adsorption rate constants 2–10× higher than WT D29 on resistant strains

Step 11 — One-step growth curve for top 3 variants

• Method: Synchronize infection of each target strain at MOI 5; wash away unadsorbed phage; resuspend in fresh media; sample every 10 min for 90 min; titer each sample by plaque assay; calculate latent period, rise period, and burst size
• Automation: BioshakeD3000 for temperature-controlled mixing; Echo525 for timed sampling transfers; Inheco Plate Incubator for constant 37°C
• Microplate: 96-v-eppendorf-951033502-deep for growth curve samples
• Expected result: Top variants show burst sizes within 2× of WT D29 on mc²155, confirming no major replication fitness cost

Step 12 — Whole-genome sequencing of top variants

• Method: Submit phage genomic DNA of top 3–5 variants for Illumina sequencing; align reads to D29 reference genome (GenBank accession AF022214); identify all mutations relative to WT; confirm tail spike variants match designed library sequences
• Automation: External sequencing service; bioinformatic analysis computational
• Expected result: Full mutation maps for top variants; confirmation that tail spike substitutions are responsible for host range expansion

Step 13 — qPCR validation of tail spike gene expression in top variants

• Method: Infect mc²155 with top 3 variants; extract total RNA at 5, 15, and 30 min post-infection; reverse transcribe; run qPCR with primers flanking the engineered tail spike region; compare expression to WT D29 tail spike
• Automation: CFX Opus qPCR machine; ATC Thermal Cycler for RT-PCR; Echo525 for qPCR reaction setup
• Microplate: 384-pcr-eppendorf-9510207XX for qPCR reactions
• Expected result: Tail spike gene expression confirmed in all top variants; no significant transcriptional defects introduced by library substitutions

Step 14 — Data analysis and fitness landscape construction

• Method: Compile EOP, adsorption rate, burst size, and sequence data for all variants; calculate fitness scores; generate heatmap of tail spike residue substitutions vs. host-range fitness; compare enriched positions to Huss et al. T7 microgravity-selected residues
• Automation: Computational (Python/R analysis)
• Expected result: Interpretable fitness landscape of D29 tail spike host-range residues; identification of convergent vs. divergent positions relative to T7 microgravity data

Step 15 — Validation experiment: PCR + Sanger sequencing of final top clone

• Method: PCR-amplify the full tail spike coding region of the top-performing D29 variant using flanking primers; run on 1% agarose gel for size confirmation; submit for Sanger sequencing; confirm exact amino acid substitutions
• Automation: ATC Thermal Cycler; Spark Plate Reader for gel quantification
• Microplate: 96-Armadillo-PCR-AB2396X for PCR setup
• Expected result: Confirmed sequence identity of top variant tail spike; complete project validation

Project Techniques

• DNA design and synthesis — Combinatorial tail spike library design; whole-plasmid synthesis via Twist Bioscience
• Recombineering / in-cell recombination — pJV53-mediated Che9c recombinase system in M. smegmatis mc²155
• Combinatorial library synthesis — Twist oligo pool-based tail spike variant library
• Plaque assay — Primary host range screening methodology
• PCR and gel electrophoresis — Colony PCR confirmation; validation sequencing
• qPCR — Tail spike gene expression validation (CFX Opus)
• Microplate automation — Full Ginkgo Bioworks automation pipeline
• Bioinformatics / sequence analysis — AlphaFold2 structural modeling; fitness landscape analysis
• Liquid handling robotics — Echo525, Multiflo, Tempest, Floi8
• Plate reader assays — Spark (OD600 growth curves), PHERAstar FSX (titer confirmation)

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