For our project, we created an mRNA editing system using CRISPR technology and editing enzymes. In making this system, we contributed 16 parts to this years registry parts number BBa_K2083000 - BBa_K2083015. These parts included all of the reporters that we designed,the editing enzymes with 1-3 repeats of an XTEN linker, and the guide RNAs for our reporters.
Out of the 16 parts we submitted, our best characterized part was BBa_K2083010. BBa_K2083010 was one of the uniquely mutated eGFP reporter proteins. In addition to a 5' untranslated region, designed to be the target binding site of the dCas9 protein, we designed the protein to have a mutated start codon. The normal start codon, that was included in the "wild type", was ATG. Our mutant had an ACG start codon. The ACG eGFP mutant was tested using fluorescence microscopy and was further confirmed using flow cytometry as part of a collaboration with the Boston University Wetlab team. Both of these techniques showed that the ACG GFP mutant had a significantly lower level of fluorescence, indicating a significantly decreased level of expression of the mutant gene.
