Team:Korea U Seoul/Project/sub desc

Enzyme purification is costly, and purified enzymes cannot be used multiple times due to decrease in activity after few reactions. To overcome this problem, ‘whole-cell biocatalysis’, which uses the microorganism itself as a catalyst, was introduced. Related to this, ‘Cell surface display’ can provide higher rate of access to the substrate, but due to the possibility of toxicity, its result has a low rate of actual expression rate. To make more efficient and stable surface display system,“sortase-based surface display at gram-positive bacteria” has been researched in many fields lately. Sortase functions as ‘protein ligase’ that recognizes specific amino acids sequence(LPXT) of each peptides and links them together. It is also associated with cell wall-bound protein and pili synthesis. ‘Sortase-based surface display’ can induce stable enzyme surface display by reducing cell toxicity and holding enzymes tightly to the cell wall with covalent bond.
However, it cannot overcome low display yield and it should include an extra step of treating sortase in vitro. Therefore, referring to sortase-based study, we are going to make enzyme polymer (enzyme pili) on cell wall by modifying pili units used in pili synthesis in other species in Corenebacterium genus. This study provides more efficient way of enzymatic process and application of pili biosynthesis

Our Team project is making pearl by E.coli induced Nacrein gene. At first, amplify kinds of nacrein genes by DH5a. Next, transformation of these genes to BW25113, BL21(DE3). Then nacrein genes make nacrein proteins. Finally they make nacrein aragonite structures itself and they forms the pearl.

Nacrein has carbonic anhydrase domain that can make CO2 to carbonic acid, and it changes to bicarbonate ion(HCO3-) and H+. And We use Nacrein, Nacrein CA, Nacrein DR, Nacrein R gene to check what gene can make lots of pearl. (CA, DR and R indicate first carbonic anhydrase domain clone, repeat deleted-clone and only repeat clone, respectively.) We can make pearl with reducing CO2.

The Korea_U_Seoul team aims to design Pearl-coli that is E.coli able to convert atmospheric CO2 into a pearl powder material. The design is based on cell surface display of nacrein in E.coli. Nacrein is a major protein component in nacre (an organic-inorganic composite layer found in outer coating of pearls). We divided nacrein into several functional regions - carbonic anhydrase (CA), calcium binding and scaffold repeats. Carbonic anhydrase domain fixes CO2 into carbonic acid changing to bicarbonate ion in aqueous solution. We assume that the other regions except CA is related to formation of nacre layer in a pearl. The whole nacrein and those functional regions (CA, NA, R) are displayed in cell surface of E.coli. We will examine if displayed nacrein in E.coli can make a pearl powder in a solution or fabricate a nacre-like structure while atmospheric CO2 is fixed into bicarbonate. Once a nacre material can be prepared from Pearl-coli, we will grow E.coli in a confined container to make synthetic pearl. The Pearl-coli has dual-function such as (1) mitigate the global warming by CO2 reduction, (2) prepare valuable pearl-like raw materials.