61. Insert Clone Selection by Sorting GFP-Expressing E. coli 

Juno Choe and Ger van den Engh 
Department of Molecular Biotechnology, University of Washington, WA 98195 
choe@biotech.washington.edu 

At a previous DOE contractors meeting (Santa Fe, 1995), we proposed a method for insert clone selection by sorting Green Fluorescent Protein-containing E. coli into individual culture wells. Direct selection of insert-containing bacteria with a cell sorter abolishes the need for clone picking. We are now using this approach in an automated, integrated process for large-fragment DNA subcloning. 

The process utilizes a vector in which insertion of DNA at a cloning site causes GFP expression. Insert-containing bacteria are selected in a cell sorter and deposited into 10 microliter wells. The vector may be amplified either by culturing the bacteria or by PCR. The amplified product may be used as a template for fluorescent dye-based sequencing reactions. Amplification by PCR avoids the need for a DNA extraction step. 

We have now demonstrated proof-of-principle for several steps of the process. We have created a vector with a GFP gene downstream of a strongly regulated promoter. The plasmid contains a Lac repressor gene. Insertion of a DNA fragment inside the repressor gene disrupts its function, resulting in GFP expression. We currently have vector strains with Blue as well as Green Fluorescent Proteins. The green protein offers the best signal to noise ratio of the two. Individual bacterial clones containing inserts can easily be detected and sorted. Results of insert amplification by PCR from single-sorted bacteria will be presented.