DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

12. A Fluorescent Sequencing Vector for High-Throughput Clone Selection by Cell Sorting

Juno Choe and Ger van den Engh

Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195-7730

engh@biotech.washington.edu

With the advent of automated high-throughput DNA sequencers, clone selection and preparation is rapidly becoming a major bottleneck in genome sequencing. We are developing an integrated clone-selection / clone-preparation process that has the potential to dramatically increase the speed of sample production. The process makes use of a sequencing vector that contains fluorescent proteins so that insert-containing bacteria can be selected with a cell sorter. The cell sorter will deposit individual bacteria onto a carrier ribbon that moves the samples in a linear procession along processing stations.

We have constructed two vectors that can be use din this process. Most recently we developed a vector containing a tandem of Blue and Green Fluorescent Proteins separated by a cloning site. This vector indicates integration of a cloned insert through the shifting of ratios of two fluorescent proteins. In the native pBGFP, a fusion protein composed of Blue Fluorescent Protein (BFP2, Clontech) and Green Fluorescent Protein (GFPmut3.1, Clontech) genes is expressed at high levels. The BFP/GFP fusion protein can be excited by UV light in the 350-400 nm range. This causes excitation of the BFP, followed by GFP due to fluorescence resonance energy transfer (FRET). When an insert is successfully ligated into the cloning site in the linker region between BFP and GFP, there is a loss of function of the GFP portion of the protein. In this case, increased BFP fluorescence will be observed with loss of observable green fluorescence. We can quantitate the ratios of these two fluorescent proteins very accurately by flow cytometry. This provides the ability to rapidly sort individual bacteria with high BFP and low GFP content at a rate of up to 10,000 per hour. Amplification of cloned inserts can be achieved by growing bacteria in culture medium and/or PCR amplification.

Under actual test conditions, ligation of fragments between 2.3-5.6 kb resulted in clear separations of two populations of E. Coli grown in liquid media: BFP/GFP expressing bacteria containing native pBGFP plasmid and BFP expressing bacteria containing pBGFP with cloned insert. Furthermore, bacteria were observed under UV fluorescence microscopy. Two clearly distinguishable phenotypes appearing either green or blue were observed.