Martin, Sonya – Fluorescent Methods for the study of Bicoid’s DNA Binding Domain

Morphogens are molecules that can aid in cell differentiation, an important process for embryo development. In the Drosophila melanogaster embryo, the Bicoid (Bcd) protein acts as a morphogen and forms an exponential concentration gradient along the anterior-posterior axis. Bicoid exerts its effect by acting as a transcription factor for important marker genes involved in pattern development in D. melanogaster. The goal of this project is to develop a fluorescent mutant of the DNA binding domain, or homeodomain, of Bcd to use in future single particle detection experiments to better understand the Bcd – DNA interaction.

Two fluorescent labelling strategies were reviewed. The first option considered was to use the green fluorescent protein (GFP). Some of the previous successful cloning and purification methods to obtain a Bcd-GFP protein fusion were reviewed. The second option considered was to attach an organic fluorophore to a cysteine in the protein. However, a point mutant to the Bcd homeodomain is needed because the wild type protein does not contain cysteine. The structure of the Bcd homeodomain was examined and possible positions for the mutation, that avoid disrupting the structure of Bcd, were proposed.

In the end, labeling the protein with a bright organic fluorophore was judged to be both more straightforward and more likely to result in a strongly fluorescent functional Bcd. Obtaining the fluorescent protein will aid in future experimentations to better understand the properties of Bcd’s DNA binding domain. This research can then be applied to better understanding Bcd’s role in embryo development.