Validated Part/Validated Contribution
Our team successfully developed a set of eGFP reporter proteins that were cloned into biobricks. The reporters were designed to determine if the APOBEC enzyme successfully edited its target. The first is a normal eGFP plasmid with an added 5' untranslated region, which was used as a target site for our CRISPR/dCas9 based editing system. The other eGFP reporter also contained a 5' untranslated region and also had a non-functional ACG start codon. The data gathered was analyzed using fluorescence microscopy and image analysis. The results of this analysis can be found here. We then confirmed our analysis by using flow cytometry as part of a collaboration with the Boston University Team
Collaboration
Our team collaborated with the Boston University Wetlab team in two major ways. First BU helped us validate both our eGFP reporter proteins, which were used as reporters for the APOBEC enzyme, with their flow cytometry machine. The flow cytometry data collected from this collaboration also functioned to validate our microscopy-based analysis system. We, in turn, helped the them using our fluorescent microscope. We analyzed the fluorescence of several of their reporter proteins to confirm their findings. See our collaboration information here.