Team:TU Delft/project/hydrocarbon tolerance


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Solvent Tolerance


To transform Escherichia coli K12 and functionally express the protein prefoldin, which confers solvent resistance.

Proposed Method

Based on: Okochi M., Kanie K., Kurimoto M., Yohda M. and Honda H.. Over expression of prefoldin from the hyperthermophilic arechaeum Pyrococus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance. Appl. Microbiol. Biotechnol. 79:443-449 (2008)

This sub-project will be the insertion and functional expression of a gene that confers solvent tolerance to Escherichia coli K12. All Escherichia coli strains are hydrocarbon sensitive, thus in order to make Escherichia coli cells capable of living on hydrocarbons they need to synthesize proteins that can make them resistant. Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. In this way it confers E.coli cells with resistance to hydrocarbons.

The sequences PhPFD-alpha, PhPFD-beta, appropriate RBS’s and promoters will be required. The selection criterion for RBS and Promoter for each construction will depend on the results obtained during the RBS characterization protocol. Tentatively, the most used promoter will be BBa_J23109, which gives a medium to low transcription level and the most used RBS will be BBa_B0032 which gives a medium translation level.

Step 1: Formation of Prefoldin BioBrick

Aim: Creation of a BioBrick for the expression of both the alpha and beta subunits of prefoldin. A medium-transcription level promoter as well as a medium-translation level ribosomal binding site is utilized.

Step 2: Characterization


  • Prefoldin: E.coli K12/407C
  • Negative control: E.coli K12
  • Positive control: Pseudomonas putida OCT

Characterization of E. coli K12/407C will involve its culturing on M9-modified liquid medium containing varying levels of cyclohexane (0%, 4%, 8%, 12% v/v). The positive control will be a colony of the P. putida OCT strain and the negative control will be an E.coli K12 colony, both grown under the same conditions. OD600 will be determined at various intervals between inoculation and 72 hours thereafter. Using these measurements the growth-related properties of each strain can be determined and analyzed accordingly.