title: A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors.
abstract: BACKGROUND
  Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The construction of these biosensors requires the genetic insertion of a judiciously chosen molecular recognition element between two distinct hues of FP. When the molecular recognition element interacts with the analyte of interest and undergoes a conformational change, the ratiometric emission of the construct is altered due to a change in the FRET efficiency. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility.
  
  
  RESULTS
  To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). This effort involved the construction and screening of 3 distinct libraries: a domain library that included several engineered binding domains isolated by phage-display; a lower-resolution linker library; and a higher-resolution linker library.
  
  
  CONCLUSIONS
  Application of this library screening methodology led to the identification of an optimized H3K27-trimethylation biosensor that exhibited an emission ratio change (66%) that was 2.3 × improved relative to that of the initially constructed biosensor (29%).
authors.affiliation: Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ibraheem
authors.forename: Hongkin
authors.initials: H
authors.lastname: Yap
authors.forename: Yidan
authors.initials: Y
authors.lastname: Ding
authors.forename: Robert E
authors.initials: RE
authors.lastname: Campbell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D057166:High-Throughput Screening Assays; D006657:Histones / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D054852:Small Molecule Libraries
chemical_list: D005456:Fluorescent Dyes; D006657:Histones; D015335:Molecular Probes; D019151:Peptide Library; D054852:Small Molecule Libraries; D008239:Lysine
references.citation: Chembiochem. 2006 Aug;7(8):1147-50
references.pmid: 16810655
references.citation: Appl Environ Microbiol. 2007 Nov;73(22):7408-14
references.pmid: 17890334
references.citation: Structure. 2007 May;15(5):515-23
references.pmid: 17502097
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10554-9
references.pmid: 15247428
references.citation: J Mol Biol. 2005 Dec 2;354(3):546-55
references.pmid: 16257413
references.citation: Genes Dev. 2003 Aug 1;17(15):1823-8
references.pmid: 12897052
references.citation: Protein Eng. 1993 Nov;6(8):989-95
references.pmid: 8309948
references.citation: Nature. 1997 Aug 28;388(6645):882-7
references.pmid: 9278050
references.citation: Anal Chem. 2009 Aug 1;81(15):5972-9
references.pmid: 19552419
references.citation: Protein Sci. 2005 Sep;14(9):2304-14
references.pmid: 16131659
references.citation: Curr Opin Chem Biol. 2010 Feb;14(1):30-6
references.pmid: 19913453
references.citation: J Mol Biol. 2006 May 26;359(1):86-96
references.pmid: 16603186
references.citation: J Am Chem Soc. 2004 May 19;126(19):5982-3
references.pmid: 15137760
references.citation: Biochem J. 2007 Oct 1;407(1):69-77
references.pmid: 17516914
references.citation: Protein Eng. 1999 Jul;12(7):613-21
references.pmid: 10436088
references.citation: Nat Protoc. 2006;1(1):16-22
references.pmid: 17406207
references.citation: Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4411-6
references.pmid: 18332427
references.citation: Methods Mol Biol. 2003;205:1-18
references.pmid: 12491876
references.citation: Anal Biochem. 2005 Jul 15;342(2):287-91
references.pmid: 15935324
references.citation: Mutat Res. 2008 Dec 1;647(1-2):21-9
references.pmid: 18723033
references.citation: Cell. 2007 Sep 21;130(6):1083-94
references.pmid: 17825402
references.citation: J Biol Chem. 2001 Aug 3;276(31):29188-94
references.pmid: 11387331
references.citation: Proc Natl Acad Sci U S A. 1983 Jan;80(1):21-5
references.pmid: 6337371
references.citation: Nat Struct Biol. 2003 Mar;10(3):187-96
references.pmid: 12567185
references.citation: Biotechniques. 2007 Nov;43(5):602, 604, 606 passim
references.pmid: 18072589
references.citation: Biochem J. 2006 Dec 15;400(3):531-40
references.pmid: 16859491
references.citation: J Bacteriol. 1995 Mar;177(6):1497-504
references.pmid: 7883706
references.citation: ACS Chem Biol. 2010 Feb 19;5(2):215-22
references.pmid: 20047338
references.citation: Genes Dev. 2003 Aug 1;17(15):1870-81
references.pmid: 12897054
references.citation: Nat Chem Biol. 2008 Oct;4(10):590-7
references.pmid: 18800048
references.citation: ACS Chem Biol. 2011 Jul 15;6(7):685-91
references.pmid: 21506563
references.citation: EMBO Rep. 2006 Apr;7(4):397-403
references.pmid: 16415788
references.citation: J Biol Chem. 2011 Jan 7;286(1):521-9
references.pmid: 21047797
references.citation: Mol Cell Biol. 2006 Apr;26(7):2560-9
references.pmid: 16537902
references.citation: Anal Biochem. 2005 Jul 1;342(1):86-92
references.pmid: 15958184
references.citation: J Bacteriol. 1995 Jul;177(14):4121-30
references.pmid: 7608087
references.citation: Mol Cell. 2003 Dec;12(6):1577-89
references.pmid: 14690609