All of the undergraduates executed all of the experiments. Each individual partook in creating the three plasmids necessary for the successful incorporation of the non-canonical amino acid. For the initial experiment, everyone helped demonstrate the localization of eGFP within the EutS compartments in various expression levels through molecular cloning. This was done in order to help improve the success rate of EutS compartmentalization. After this was proven, subteams were formed.
Jonah, Brandon, Maxwell, and Jessica:
Their primary tasks were to create the desired tri-plasmid system; this included the EutS shell protein, EutC tagged with eGFP, and the pEVOL plasmid.
Elizabeth and Will:
In order to successfully incorporate the non-canonical amino acid into this tri-plasmid system, genomic edits on the EutS genome must be made. Positions for mutagenesis was done in various spots on the genome in order to test whether or not the incorporation facilitated with the formation of the shell protein.Their primary tasks were to create a library of mutants with MUSE using CRISPR-Cas9. By making genomic edits, the native EutS shell protein was activated; also, variants created by these edits were used to test against the native EUTS compartment.
Michael Donovan:
Used PyRosetta position scoring to find possible binding sites of assembled hexamers. Looked at locations at the edge of EutS hexamer when aligned to E.coli EutM.
Co-Advisers
Brian DeDecker & Robin Dowell:
Handled the finances and written documents for the iGem team at CU Boulder.
Mentors
Michael Brasino:
Oversaw the project on a week-to-week basis, provided insight on how to approach mishaps and guided us in the right direction for the experiments. He also helped create the azobenzene.
Marcelo Bassalo:
Helped design the genomic Eut operon constructs to test the native system and generate the library variants. He provided the muse CREATE system using CRISPR-Cas9 to conduct the experiments.
Peter Otoupal:
Helped coordinate funding and materials. Also participated in idea generation.
Sponsors
Muse:
Generated a library of EutS variants
New England Biolabs:
Materials
Integrated DNA technologies:
Funding for oligomer purchases
Biofrontiers:
Funding
IQ biology Program:
Funding
Molecular Cellular and Developmental Biology Program:
