Team:TU Delft/project/hydrocarbon tolerance

From 2010.igem.org

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(Step 1: Formation of Prefoldin BioBrick)
(Step 1: Formation of Prefoldin BioBrick)
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Check the [http://mrgene.com/desktopdefault.aspx/tabid-2/?gclid=CK_v3NyTtqMCFUl_3god1zv0cQ Mr. Gene] sequences by clicking on the links: [http://2010.igem.org/wiki/images/b/b5/PhPFDa.gb Prefoldin alpha] and [http://2010.igem.org/wiki/images/2/26/PhPFDb.gb Prefoldin beta]
Check the [http://mrgene.com/desktopdefault.aspx/tabid-2/?gclid=CK_v3NyTtqMCFUl_3god1zv0cQ Mr. Gene] sequences by clicking on the links: [http://2010.igem.org/wiki/images/b/b5/PhPFDa.gb Prefoldin alpha] and [http://2010.igem.org/wiki/images/2/26/PhPFDb.gb Prefoldin beta]
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''In silico'' constructions were made, and you can check below our final construct, [http://serialbasics.free.fr/Serial_Cloner.html SerialCloner] was used for this purpose.
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''In silico'' constructions were made, and you can check below our final construct; the image displayed was generated using [http://serialbasics.free.fr/Serial_Cloner.html Serial Cloner 2.1] .
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Revision as of 09:54, 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.

Before any design, we checked on literature about reports related to solvent/hydrocarbon tolerance. The paper published by Okochi and co-workers caught our attention up, according to their paper E. coli expressing prefoldin from Pyrococcus horikoshii OT3 grows quite well in the presence of high concentrations (10%v/v) of cyclohexane. Cyclohexane causes membrane leaking and other toxic effects, which makes it a good candidate for testing solvent/hydrocarbon tolerance.

The original prefoldin alpha and prefoldin beta DNA sequences differ in composition from the E. coli sequences and also contained some undesired restriction sites, that's why we decided to enhance our sequence for a successful expression on E. coli, for that purpose we used jcat website. Once we had the enhanced sequence, we added the standard biobrick prefix and suffix and couple of weeks later our dear Mr. Gene sent us back the sequences in a pANY plasmid.

Check the Mr. Gene sequences by clicking on the links: Prefoldin alpha and Prefoldin beta

In silico constructions were made, and you can check below our final construct; the image displayed was generated using Serial Cloner 2.1 .

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 available here!

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.