Part:BBa_K398406

Figure 1 - Proteins [http://www.rcsb.org/pdb/explore/explore.do?pdbId=2ZDI phPFDα and phPFDβ]

Solvent tolerance cluster

BBa_K398406 enables E.coli of working in two-phase systems with resistance to high concentrations of hydrophobic molecules in the membrane. A growth rate increase of 62% in comparison to a standard E.coli was achieved at concentrations of 8% (v/v) n-hexane.

Introduction

The effect of organic solvents to organisms can be detrimental: they can accumulate in the cell membrane changing the composition, thus affecting its physiological function. BBa_K398406 produces prefoldin, a protein 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. The prefoldin constists of two subunits: phPFDα and phPFDβ (Figure 1), two alpha subunits bind to four beta subunits and the produce the biologically functional Prefoldin. Prefoldin increases the tolerance to alkanes by acting as chaperones, which correct protein misfolding /unfolding in the presence of alkanes.

Characterization

The tolerance of our bacteria to organic solvent and salt was tested by studying its growth under varying conditions. The solvent tolerance was tested under varying concentrations of cyclo-hexane, 10%(v/v) octane-hexane mixture and n-hexane, biomass production was monitored by measuring the absorbance at 595 nm in M9 medium. The [http://2010.igem.org/Team:TU_Delft#page=Project/tolerance characterization of BBa_K398406] has been further described on the TU Delft iGEM Team 2010 wiki.

Results

As can be seen from figure 2, the BBa_K398406 increases the tolerance of E.coli K12 to 8% (v/v) n-hexane with 62% compared to the native E.coli strain.

Figure 1: Model of the Solvent tolerance

Figure 2:Comparison of the growth rates between E. coli K12 and E. coli 406A at different n-hexane concentrations in M9 medium.

References

M. Okochi, K. Kanie, M. Kurimoto ,M. Yohda & Hiroyuki Honda Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance Appl Microbiol Biotechnol 79:443–449 (2008)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 37
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 37
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 37
  
• 1000
  COMPATIBLE WITH RFC[1000]