Experiments

We first did paper research, learned about the basic knowledge about Nonketotic Hyperglycinemia, and brainstormed about how we can develop a design to help the patients. In a particular paper, we found a method of carrying out the phenylalanine-to-cinnamaldehyde metabolic pathway in modified E. coli and learned the method. We bought particular genes including SmPAL, ScCCL, and AtCCR from IDT and added them into E. coli cells that we have.

In this process, we used the following materials:

E. coli K-12

Standard bacterial growth media and equipment

DNA Ligation Kit (New England Biolabs)

Plasmid Isolation Kit (New England Biolabs)

Restriction enzymes (New England Biolabs)

Agarose

Gel electrophoresis buffer

SDS-PAGE Premade Gels (Bio-Rad)

*note that the bacteria used in the experiments are under biosafety level 1

We went through digestion to cut the DNA in the E. coli. We went through ligation and transformation to link the added gene with E. coli’s original gene. To test if we successfully added the gene into the E. coli cells, we first picked colonies, and then we went through plasmid preparation and PCR confirmation to get ready for gel electrophoresis. After we observed the size of the DNA through gel electrophoresis and ensured that the genes were added into the E. coli cells, we went through cryopreservation to store some of our edited E. coli away. Later, we worked on adding the stop codon onto the gene we added into E. coli, and went through the cycle of digestion, ligation, transformation, pick colonies, plasmid preparation, PCR confirmation, and gel electrophoresis several times.