Jason Brown

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Synchronization and Media Exchange in Large
ScaleCaenorhabditis elegans Cultures
CIB Fermentation Lab Jason Brown

• Jason Brown
• Biological and Irrigation Engineering
• MS Project

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• Outline
• Intro to C. elegans
• General
• Molecule of interest
• Produce 100L synchronous culture
• Culture issues
• Scale up issues
• Synchronization
• Conditioned media production
• Approach to study
• Synchronization apparatus
• Filtration apparatus

http//info.med.yale.edu/mbb/koelle/
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• C. elegans
• Species of nematode (round worm)
• Model organism
• Study of sex attraction
• Growth stages
• Embryo, L1, L2, L3, L4, adult
• Dauer stage (alternate stage of dormancy)

http//www.wormatlas.org/handbook/anatomyintro/ana
tomyintro.htm
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• Nervous system research
• Sensory pathway
• Male attractant
• Putative hermaphrodite pheromones
• Male neurons distinct from hermaphrodites

http//www.molbio.unizh.ch/hengartner/research/neu
robiology/index.htm
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• Issues with obtaining molecule
• Potent but produced in macro mole quantities
• Culture 10L for one NMR analysis
• 100L needed for consecutive analysis
• Simplify analysis
• Synchronous culture
• Same growth stage
• Conditioned media

CIB Fermentation Lab Jason Brown
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• Produce 100L
• Culture issues
• Conditions that induce dauer state
• Temp
• Food
• Nutrients
• Population density
• Modify reactor
• Meet conditions
• Reduce stress

CIB Fermentation Lab Jason Brown
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• Scale up Issues
• Synchronization
• Tedious and precise procedure
• Conditioned media production
• Media exchange
• Minimal stress to worms
• Remove conflicting compounds

http//lifesci.rutgers.edu/grantlab/frames.html
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• Approach to Issues
• Scale up synchronization
• Tangential flow filtration
• Nylon mesh
• Retain eggs
• Scale up media exchange
• Adult retention Filter
• Nylon mesh
• Retain adults

CIB Fermentation Lab Jason Brown
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• Current progress
• Modifying reactors
• Impellers
• Filter apparatus
• Continuous feed
• Synchronization
• Small scale prototype
• Scale-up prototype
• Modify apparatus
• Test apparatus

CIB Fermentation Lab Jason Brown
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• Current Progress
• 5L culture
• 5,000/ml
• Synchronous
• Conditioned media
• Prototypes tested
• Redesign

CIB Fermentation Lab Jason Brown
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• Outcome of research
• Mass synchronous C. elegans cultivation
• Future scientific advances and discoveries
• Putative pheromone analysis
• Proteomics research
• Mass cultivation transgenic worms
• Agricultural and industrial uses
• Pest management

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• Acknowledgements and collaboration
• The Center for Integrated BioSystems (USU)
• Temperature control equipment
• 5-100L bioreactors
• Filtration equipment
• Mark Signs (CIB)
• Bioreactor modification
• Design input
• Jorgensen Lab (University of Utah)
• Funding
• Dr. Jamie White (UofU)
• Analyze male attractant pheromones
• C. elegans small scale culture methods
• Synchronization
• Conditioned media production

http//logo.cafepress.com/3/521462.139973.jpg
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Thank You. Questions?
http//www.genetics.wustl.edu/tslab/

• References
• Bargmann CI Chemosensation in C. elegans. The
  C. elegans Research Community, WormBook October
  25, 2006, doi/10.1895/wormbook.1.123.1,
  http//www.wormbook.org.
• Butcher RA, Fujita M, Schroeder FC, Clardy J
  Small-molecule pheromones that control dauer
  development in Caenorhabditis elegans. Nature
  Chemical Biology 2007, 3.7420-422
  doi10.1038/nchembio.2007.3.
• Fabian TJ, Johnson TE Production of
  age-synchronous mass cultures of Caenorhabditis
  elegans. Journal of Gerontology Biological
  Sciences 1994, 49.4B145-B156.
• Lewis JA, Fleming JT Basic culture methods
  Life cycle of Caenorhabditis elegans. In Methods
  in Cell Biology. Volume 48. Academic Press
  19953-29.
• Schroeder FC Small molecule signaling in
  Caenorhabditis elegans. ACS Chemical Biology
  2006, 1.4198-200 doi10.1021/cb600173t
• Shaham, S., ed., WormBook Methods in Cell
  Biology. The C. elegans Research Community,
  WormBook January 02 2006, doi/10.1895/wormbook.1.4
  9.1, http//www.wormbook.org.
• Shuler M.L, Kargi F Scale-up. In Bioprocess
  Engineering Basic Concepts. 2nd Edition.
  Prentice Hall PTR 2002 297-301.
• White JQ Personal communications, 2007.
• White JQ, Nicholas TJ, Gritton J, Truong L,
  Davidson ER, Jorgensen EM The sensory circuitry
  for sexual attraction in C. elegans males.
  Current Biology 2007, 17.211-11, Suppl
  17.211-7, doi10.1016/j.cub.2007.09.011