Optical Processing of Bacterial Libraries for Directed Evolution

Allan Wallace Scruggs, Neal Woodbury

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


Selection of phenotypically distinct bacterial colonies on a Petri dish is typically performed by one of two methods: chemical or mechanical. Chemical methods (e.g., antibiotic selection) rely on inherent growth advantages of the unique phenotypes desired and thus have limited applicability. Mechanical methods are generally slow and require relatively large colonies (typically hundreds of colonies per plate). Here the use of imaged light to select bacterial colonies is explored, employing either photodynamic therapy agents or a ferrochelatase mutation in combination with porphyrin precursors to sensitize the bacteria to light and a computer-controlled light projection system to illuminate some bacterial colonies while leaving others in the dark. A CCD camera was used to distinguish between bacteria expressing green fluorescent protein (GFP) from nonfluorescent colonies. The fluorescence image from the camera was then used to create a virtual masking image for photoselection. Using a simple commercial projector it was possible to confer a 56-fold selective advantage to colonies expressing GFP. This represents a potentially powerful tool in directed evolution experiments using large libraries.

Original languageEnglish (US)
Pages (from-to)445-451
Number of pages7
JournalBiotechnology and bioengineering
Issue number4
StatePublished - Nov 20 2003


  • Directed evolution
  • Light sensitive
  • Photodynamic treatment
  • Selection

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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