NSF Educator Workshop in Cell-free Biomanufacturing

Workshop Summary

Synthetic biology and biomanufacturing are buzzwords of an emerging bioeconomy based on our increasing ability to manipulate living systems. This workshop will equip bioscience educators with the knowledge, skills, and resources to introduce students to a simple workflow to manufacture bacteriophages (phages) with new host specificity, illustrating how biomolecules are adapted to interact with cell-surface receptors.

Join Vincent Noireaux, an instructor of the annual Synthetic Biology course at Cold Spring Harbor Laboratory, to come up-to-the minute with a new, cell-free transcription-translation (TXTL) system he developed to biomanufacture phages in vitro. This workshop emerges from a distinguished lineage of bacteriophage research that began at Cold Spring Harbor Laboratory (CSHL) and provided the first tools to explore the molecular mechanics of living cells. The “Phage Course,” founded at CSHL in 1945 by Max Delbrück and Salvador Luria, trained the first two generations of molecular biologists. Al Hershey and Martha Chase’s “blender experiment,” conducted at CSHL in 1952, provided conclusive evidence that DNA is the molecule of heredity. Delbrück, Luria, Hershey shared the 1969 Nobel Prize for this seminal work.

Bacteriophages are being rediscovered as powerful tools to meet the pharmaceutical challenge of untreatable, multi-drug resistant bacterial infections. In the food industry, bacteriophages prevent formation of biofilms on equipment surfaces, sanitize fresh fruits and vegetables, and extend the shelf life of packaged foods. Using phages to treat bacterial infections in livestock increases resilience and helps eliminate the reservoir of antibiotic-resistant bacteria. Phages also provide a virtually limitless source of bioactive materials that are increasingly exploited in biotechnology, nanotechnology, and bioremediation.

Phages are used in education as examples of simple genetic systems. The SEA-PHAGES Program of the Howard Hughes Medical Institute annotates phage genomes, and is one of the most widely implemented infrastructures for course-based undergraduate research experiences (CUREs). TXTL is a logical next step for students who have been exposed to phage and/or bacterial genetics, providing them an opportunity to explore the use of phages in biomanufacturing.

Workshop participants will conduct hands-on experiments to express reporter genes and whole phage genomes in vitro, using a cell-free extract. Participants will complete an entire workflow to engineer wild-type T7 phage to infect a new E. coli host. This begins with long PCRs to amplify several fragments of the T7 genome, plus PCR mutagenesis of the tail fiber gene. The PCR products are then assembled and packaged as complete T7 genomes using the cell-free TXTL system. Spotting assays compare the host range of wild-type and mutant T7 phages. Participants will then conduct nanopore DNA sequencing, delivering same-day results to investigate point mutations in the tail fiber gene that account for infectivity.

A $500 stipend will be provided. Travel funds are available.

Instructors

Vincent Noireaux, Ph.D. is Professor of Synthetic Biology and Biological Physics at the University of Minnesota. His lab uses cell-free transcription-translation (TXTL) to produce proteins in vitro and to engineer biochemical systems such as gene circuits, phages, and synthetic cells. Dr. Noireaux earned a BS in Applied Physics from the University of Tours (France), his Ph.D. at the Curie Institute in Paris, and he did a postdoc at The Rockefeller University in New York City. He is co-instructor of the influential Cold Spring Harbor Laboratory postgraduate training course on Synthetic Biology.

Vincent Noireaux, Ph.D.

Professor at the University of Minnesota

Steven Bowden, Ph.D. is Assistant Professor of Food Safety Microbiology at the University of Minnesota. His lab studies foodborne pathogens such as Salmonella, Listeria monocytogenes, and Escherichia coli, and the use of bacteriophages to detect and control these bacteria. Dr. Bowden earned a BSc in Biochemistry from the University of Birmingham (UK) and his Ph.D. at the University of Cambridge (UK). He held postdoctoral positions at the Quadram Institute (UK), University of Cambridge (UK), Nara Institute of Science and Technology (Japan), and University of Minnesota.

Steven Bowden, Ph.D.

Professor at the University of Minnesota

Anna Feitzinger, Ph.D. is Assistant Director for Science of the DNA Learning Center of Cold Spring Harbor Laboratory, a science center devoted entirely to public genetics education. She applies her expertise in quantitative biology and biochemistry to bridging the gap in bioinformatics education at the pre-college and college level. She is spearheading the use of Oxford Nanopore sequencing in education, with the dream that sequencing technology will be available to students in classrooms worldwide. She received a B.A. in Chemistry from the City University of New York, Hunter College, and a Ph.D. in Biology at University of California, Davis.

Anna Feitzinger, Ph.D.

Assistant Director for Science of the DNA Learning Center of Cold Spring Harbor Laboratory

Dave Micklos, D.Sc. is founder and Executive Director of the DNA Learning Center of Cold Spring Harbor Laboratory, which encompasses five science outreach labs in metro New York/New Jersey totaling 43,000 ft². Each year, more than 500,000 students use textbooks or commercial lab kits developed by the DNALC, analyze DNA sequences using DNALC bioinformatics tools, or do experiments at the DNALC or an allied science center. Dave is a Fellow of the American Association for the Advancement of Science, and his work has been recognized with prizes from the Genetics Society of America, Charles A. Dana Foundation, American Society for Cell Biology Award, and Science Magazine.

Dave Micklos, D.Sc.

Executive Director of the DNA Learning Center of Cold Spring Harbor Laboratory

Workshop Schedule

Download Schedule (PDF)

Monday, June 3
8:30 AM	Workshop Objectives, Concepts, and Workflows
9:00 AM	Phage T7 Genome Assembly and Tail Fiber Mutagenesis (Part 1) • Set up Long PCR Reactions (~1 hour)
11:00 AM	TXTL of Reporter Genes (Part 1) • Set up Reactions of Linear and Plasmid DNA TXTL Production of Phage T7 (Part 1) • Set up Reactions at Different Concentratio
12:30 PM	LUNCH
12:30 PM	Phage T7 Genome Assembly and Tail Fiber Mutagenesis (Part 2) • PCR Clean-up, DNA quantitation, and Gel Electrophoresis • PCR Mutagensis (~2 hours)
3:00 PM	TXTL Production of Phage T7 (Part 2) • Set up Spotting Assay and Overnight Incubation • Set up Plate Reader
4:30 PM	Phage T7 Genome Assembly and Tail Fiber Mutagenesis (Part 3) • PCR mutagenesis: Clean-up, DNA quantitation, and Gel Electrophoresis

Tuesday, June 4
8:30 AM	Phage Genome Assembly and Tail Fiber Mutagenesis (Part 4) • DNA Assembly Reactions and TXTL Reactions of T7 Wild Type and T7 Mutants
10:30 AM	TXTL of Reporter Genes (Part 2) • View Results Under UV Light TXTL Production of Phage T7 (Part 3) • Results, Calculate Plaque Forming Units (PFUs) • Results from Plate Reader
12:00 PM	LUNCH
1:00 PM	Seminar: Phage Biology, Steve Bowden
2:00 PM	Phage Genome Assembly and Tail Fiber Mutagenesis (Part 5) • Electrophoresis of T7 Genome Assemblies (optional) • Set up Spotting Assay and Overnight Incubation
4:00 PM	Questions and Answers

Wednesday, June 5
9:00 AM	Phage Genome Assembly and Tail Fiber Mutagenesis (Part 6) • Pick and PCR of Amplified Tail Fiber Mutants
10:00 AM	Seminar: Nanopore DNA Sequencing, Anna Feitzinger
11:00 AM	Phage Genome Assembly and Tail Fiber Mutagenesis (Part 7) • Gel Electrophoresis of Amplified Tail Fiber Mutants • Spoting Mutant and Wild-type Plaques
12:30 PM	LUNCH
1:00 PM	Nanopore DNA Sequencing of Tail Fiber Mutants (Part 1) • Rapid Adaptor Library Prepatation • Load Nanopore Flow Cell and Begin Sequencing • Review Early Results

Thursday, June 6
9:00 AM	Phage Genome Assembly and Tail Fiber Mutagenesis (Part 8) • Calculate Efficiency of Plating by Spotting Mutant and Wild-type Plaques
9:30 AM	Nanopore DNA Sequencing of Tail Fiber Mutants (Part 2) • Create Consensus, Align Sequences, and Analyze Mutations in Tail Fiber Sequence
11:30 AM	Classroom Implementation: Settings, Challenges, Bottlenecks, Support Post-Workshop Survey
12:30 PM	Graduation
1:00 PM	LUNCH (optional)

Workshop Location

DNA Learning Center NYC at City Tech

62 Tillary Street,
(Entrance at the corner of Tillary and Adams Streets)
Brooklyn, New York 11201

Phone: (718) 285-0389

Funded and Developed by:

Funded by grants from the National Science Foundation:
Future Manufacturing #2228971; Improving Undergraduate STEM Education #1821657

Using Cell-free Systems to Synthesize and Express Bacteriophage Genomes:

Practical Biomanufacturing for Bioscience Educators

June 3–6, 2024
DNA Learning Center NYC at City Tech
62 Tillary Street, Brooklyn, New York 11201

Workshop Summary