Team:Northeastern Boston/Design

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EXPRESSION PLASMID

BBa_K1857002Protein Expression Plasmid

The goal of Northeastern's 2015 team was to standardize heterologous protein production. Chlamydomonas reinhardtii have attracted significant attention for their potential use in the production of therapeutic proteins, and this should be further explored and developed. [1, 2, 3]

C. reinhardtii are capable of complex protein production, with di-sulfide bonds and glycosylation patterns similar to mammalian cells. [4] Furthermore, their primary carbon source is CO₂ and they can be scaled cheaply to large volumes in raceway ponds. [5]

The single largest barrier to widespread C. reinhardtii adoption is the low levels of heterologous protein production. [1] Therefore, to facilitate ease of testing for nuclear protein production in C. reinhardtii, we have produced an iGEM compatible protein expression plasmid.

This plasmid is comprised of two HSP70A-RBCS2 fusion cassettes. The first HSP70A-RBCS2 promoter—with an RBCS2 intron—is flanked by the iGEM prefix and suffix. For straightforward production of a heterologous proteins in the nucleus, the suffix may be removed with SpeI and PstI and replaced with a gene of interest. The 3’UTR of RBCS2 (in effect, the terminator) is downstream of this suffix.

Alternatively, the entire HSP70A-RBCS2 promoter can be removed via EcoRI and PstI, and replaced with an alternate promoter. In this way, alternate promoters could be compared for their relative strength, for example, in the testing of the relative abundance of mVenus.

The second part of the protein expression plasmid is a Hygromycin B resistance cassette. This will allow for transformation selection with Hygromycin B.