Optical Processing of Bacterial Libraries for Directed Evolution

Allan Wallace Scruggs, Neal Woodbury

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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
Volume84
Issue number4
DOIs
StatePublished - Nov 20 2003

Fingerprint

Libraries
Green Fluorescent Proteins
Light
Bacteria
Processing
Ferrochelatase
Proteins
Projection systems
Photodynamic therapy
Porphyrins
Photochemotherapy
Antibiotics
CCD cameras
Fluorescence
Cameras
Anti-Bacterial Agents
Phenotype
Mutation
Growth
Experiments

Keywords

  • Directed evolution
  • Light sensitive
  • Photodynamic treatment
  • Selection

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology

Cite this

Optical Processing of Bacterial Libraries for Directed Evolution. / Scruggs, Allan Wallace; Woodbury, Neal.

In: Biotechnology and Bioengineering, Vol. 84, No. 4, 20.11.2003, p. 445-451.

Research output: Contribution to journalArticle

@article{60a6167fde4040a3b0f2754c3e358459,
title = "Optical Processing of Bacterial Libraries for Directed Evolution",
abstract = "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.",
keywords = "Directed evolution, Light sensitive, Photodynamic treatment, Selection",
author = "Scruggs, {Allan Wallace} and Neal Woodbury",
year = "2003",
month = "11",
day = "20",
doi = "10.1002/bit.10794",
language = "English (US)",
volume = "84",
pages = "445--451",
journal = "Biotechnology and Bioengineering",
issn = "0006-3592",
publisher = "Wiley-VCH Verlag",
number = "4",

}

TY - JOUR

T1 - Optical Processing of Bacterial Libraries for Directed Evolution

AU - Scruggs, Allan Wallace

AU - Woodbury, Neal

PY - 2003/11/20

Y1 - 2003/11/20

N2 - 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.

AB - 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.

KW - Directed evolution

KW - Light sensitive

KW - Photodynamic treatment

KW - Selection

UR - http://www.scopus.com/inward/record.url?scp=0142041422&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0142041422&partnerID=8YFLogxK

U2 - 10.1002/bit.10794

DO - 10.1002/bit.10794

M3 - Article

C2 - 14574702

AN - SCOPUS:0142041422

VL - 84

SP - 445

EP - 451

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

SN - 0006-3592

IS - 4

ER -