Difference between revisions of "Team:CU-Boulder/Collaborations"

Using the CRSPR based CREATE system designed by MUSE, we were able to extend the edits we were making to our plasmid to the genomic level. We did this by expressing the native EutSMNLK compartment in E. Coli cells by introducing an IPTG inducible promoter and then transforming the cells with two sets of genomic edit libraries, provided by MUSE. The first was a library in which each amino acid was replaced with the every other possible amino acids, one at a time. The second provided library contains the amber stop codon at each codon within the genome. With the special tRNA expressed by the Schultz plasmid, the amber stop codon can actually be used to introduce azobenzene to the compartment. The collaboration with MUSE has allowed the team to extend the range and shorten the time required for adding mutations to our system as well as will yield a comparison of the engineered EutS protein to the native EutSMNLK.

We are hopeful for mutations in the genome that create a successful nano- compartment and may improve the stability of the compartment. Ethanolamine is toxic to the cells and is metabolized in Eut complexes. To test the mutations, the two libraries will be grown on ethanolamine plates, with varying concentrations of ethanolamine. Only mutants that can successfully create the compartment will survive on these plates and, theoretically, the plasmids with a beneficial mutation will be found only on the plates with a high concentration of ethanolamine. To test which mutations survived as well as the frequency of these, we will use high throughput sequencing to verify the mutations and quantify which mutations yielded as successful compartment. These data will provide insight into the location of the genome in which mutations are favorable to form this compartment and set up further research into the use of these mutated compartments.