Team:TU Delft/project/hydrocarbon tolerance

From 2010.igem.org

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[http://2010.igem.org/wiki/images/0/04/406C.gb Genebank annotated file]
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===Step 2: Characterization===
===Step 2: Characterization===

Revision as of 09:16, 13 August 2010

Contents

Solvent Tolerance

It is known that hydrocarbons and other solvents have toxic effects on cells. Due to this fact, it is necessary to confer Escherichia coli K12 hydrocarbon/solvent tolerance, so that it can grow on biphasic systems with toxic concentrations of these compounds.

Aim

To functionally express the protein prefoldin from Pyrococcus horikoshii OT3 in Escherichia coli K12.

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)


For this part of our project, we will insert and functionally express the alpha and beta sub-units of Pyrococcus horikoshii OT3 prefoldin; this protein confers solvent/hydrocarbon tolerance to E. coli K12 cells, according to the cited literature. Prefoldin is a jellyfish-shaped hexameric chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding, this is the molecular mechanism behind solvent/hydrocarbon resistance prefoldin-associated.

PhPFD-alpha, PhPFD-beta, will be ligated to the appropriate RBS and promoter. The selection criterion for RBS and promoter 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.

406C.jpg
Feature Function
alkB2 Alkane 1-monooxygenase (Gordonia sp. TF6)
B0015 Transcriptional (double) terminator
B0042 Transcriptional terminator
B0053 Transcriptional terminator
B0054 Transcriptional terminator
B0055 Transcriptional terminator
B0062 Transcriptional terminator
G00000 Standard prefix
G00001 Standard suffix
G00100 VF2 primer binding site
G00102 VR primer binding site
I50032 p15A replication origin
J61100 RBS Anderson family
J23100 Promoter
P1005 TetR
rubA3 Rubredoxin A3 (Gordonia sp. TF6)
rubA4 Rubredoxin A4 (Gordonia sp. TF6)
rubR Rubredoxin reductase (Gordonia sp. TF6)

Genebank annotated file

Step 2: Characterization

Strains:

  • 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.