title: Fluorescent protein-based methods for on-plate screening of gene insertion.
abstract: BACKGROUND
  Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.
  
  
  RESULTS
  Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.
  
  
  CONCLUSIONS
  The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.
authors.forename: Stanley S C
authors.initials: SS
authors.lastname: Wong
authors.forename: Kevin
authors.initials: K
authors.lastname: Truong
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002147:Calmodulin / Q000737:chemistry; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D017930:Genes, Reporter; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D016254:Mutagenesis, Insertional; D009154:Mutation; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D000097573:Red Fluorescent Protein
chemical_list: D002147:Calmodulin; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein