A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein - target protein fusions.

Abstract

A simple broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E. coli transformation with no further procedures. Plating at high colony density yields fluorescent colonies. Plasmids purified from fluorescent colonies contain random, in-frame fusion proteins into the target gene. The plate screen also results in expressed, stable proteins. A large library of chimeric proteins was produced that was useful for downstream research. The effect of using different fluorescent proteins was investigated as well as the dependence of linker sequence between the target and fluorescent protein open reading frames. The utility and simplicity of the method was demonstrated by the fact that it has been employed in an undergraduate biology laboratory class without failure over dozens of class sections. This suggests that the method will be useful in high impact research at small liberal arts colleges with limited resources. However, in frame fusion proteins were obtained from eight different targets suggesting that the method is broadly applicable in any research setting. SummaryThis report describes a simple screen to obtain random insertions of fluorescent proteins into a target protein of interest. The screen results in a useful library of mutated and functional tagged proteins that can be employed to investigate protein biochemical activity, protein structure, protein function and protein cellular localization. The screen is robust, generally applicable, and has been employed in an undergraduate laboratory class. It is also useful for studies in advanced research-intensive projects carried out in institutions of all types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/578020v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@22dc94org.highwire.dtl.DTLVardef@bf789dorg.highwire.dtl.DTLVardef@87c53forg.highwire.dtl.DTLVardef@1a6c0e9_HPS_FORMAT_FIGEXP M_FIG C_FIG