Team:TU Delft/Project/tolerance


Revision as of 13:57, 10 September 2010 by Ravandervalk (Talk | contribs)


Our goal was to develop a cellular chassis capable of convert hydrophobic chemicals (alkanes) and use them as sole carbon and energy source. However, in order to have a cell capable of making these kinds of bioconversions it was necessary to think about the environment in which these cells will be growing. The two main challenges found were salinity and organic solvent toxicity.

Our cells are meant to work in tailing waters and oil-spills; a high salt concentration is expected. Due to this fact we need to confer halotolerance to our cells. Moreover, two-phase systems are difficult to work with due to the presence of high levels of organic solvents in the environment. Which is the main reason for design a solvent-tolerant cell.


Solvent tolerance

The effect of organic solvents is widely known; mostly they can accumulate in the cell membrane changing the composition, thus affecting its physiological function. Our goal was to develop an E. coli strain capable of working in two-phase systems with resistance to high concentrations of hydrophobic molecules in the membrane.

Different papers were checked, some of them reporting an increase in the organic solvent tolerance of E. coli after the over-expression of efflux-pumps, ostA E. coli gene and some other genes. Efflux-pumps seemed to complex due to de amount of genes required and because of the fact that these systems also confer antibiotic resistance to the cells; on the other hand ostA gene is a common gene in E. coli, in order to have a negative control it is required to disrupt the gene which could make complex the characterization of our biobrick.

From the work of Okochi and co-workers, we discovered the prefoldin system from Pyrococcus horikoshii OT3. Prefoldin is a part of a widely spread group of proteins: chaperones. These proteins have the property of helping during the protein folding process, and are related also to thermal shock resistance. Therefore we opted for the use of two proteins; namely phPFDα and phPFDβ. These proteins increase the tolerance to alkanes by acting as chaperones, which correct protein misfolding /unfolding in the presence of alkanes.

Salt tolerance

Salts are essential for life, but not all life requires the same amount or the same type of salts. But because of these varying conditions the salt stress differs from environment to environment. It is difficult for a species to adapt to different salt stress levels. To compensate for the cells innate vulnerability to higher salt concentrations we chose a protein from Chlamydomonas sp. W80 that increases the salt tolerance of E.Coli. This protein is called bbc1.The exact mechanism of the increased salt tolerance is as of yet unknown. But since bbc1 shows a high homology to RBS binding proteins, it is theorized that it may prevent/stabilize the folding structure of the ribosome under high salt stress conditions.