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What is the context of this research?

Typically scientists try to make proteins in a number of expression systems, each of which requires its own expression vector. It's pretty much trial and error to find out what expression system can produce a large amount of the target protein. In this project I propose to create and validate an expression vector that can express proteins in E.coli, insect cells, mammalian cells, and pichia pastoris. This vector should be IP free and able to be used by anyone, anywhere.

What is the significance of this project?

Recombinant protein production is the cornerstone of modern Biochemistry and Structural Biology. Being able to make these proteins in large amounts is often really useful for a variety of applications including structure-based drug design, immunization for vaccine and antibody discovery, and phage display. Similar vectors for three expression systems exist, but they are extremely expensive, which puts the time and cost savings provided by these vectors out of reach of the people that have the least money to spend.

What are the goals of the project?

I will use these funds to design and create the new vector, and then my clone a test set of 5 IP-free fluorescent proteins in single expression system vectors and then in the combined vector.

5 genes in E.coli

5 genes in Baculovirus infected insect cells

5 genes in mammalian

5 genes in pichia pastoris

5 genes in the multiple system expression vector



This means making a total of 25 expression constructs and testing them all in their respective systems, and then comparing all the results. These results will be published with data in an open access, peer reviewed journal. In the end the new expression plasmid will get uploaded to Addgene, which will allow anyone to download the sequence and receive a small aliquot of the plasmid so that they can use it in their own research.