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Team:TU Delft/project/hydrocarbon tolerance
From 2010.igem.org
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.
Prefoldin alpha and prefoldin 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 [http://www.ncbi.nlm.nih.gov/nuccore/14589963/?from=474271&to=474726&strand=true&report=genbank prefoldin alpha] and [http://www.ncbi.nlm.nih.gov/nuccore/14589963/?from=478012&to=478365&report=genbank 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 [http://www.jcat.de/ jcat] website. Once we had the enhanced sequence, we added the standard biobrick [http://partsregistry.org/Assembly:RBS-CDS_issues prefix] and [http://partsregistry.org/Assembly:RBS-CDS_issues suffix] and couple of weeks later our dear [http://mrgene.com/desktopdefault.aspx/tabid-2/?gclid=CK_v3NyTtqMCFUl_3god1zv0cQ Mr. Gene] sent us back the sequences in a pANY plasmid.
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] .
Check the [http://mrgene.com/desktopdefault.aspx/tabid-2/?gclid=CK_v3NyTtqMCFUl_3god1zv0cQ Mr. Gene] sequences by clicking on the links: Prefoldin alpha and Prefoldin beta
| Feature | Function |
| [http://partsregistry.org/Part:BBa_B0015 B0015] | Transcriptional (double) terminator |
| [http://partsregistry.org/Part:BBa_B0042 B0042] | Transcriptional terminator |
| [http://partsregistry.org/Part:BBa_B0053 B0053] | Transcriptional terminator |
| [http://partsregistry.org/Part:BBa_B0054 B0054] | Transcriptional terminator |
| [http://partsregistry.org/Part:BBa_B0055 B0055] | Transcriptional terminator |
| [http://partsregistry.org/Part:BBa_B0062 B0062] | Transcriptional terminator |
| [http://partsregistry.org/Part:BBa_G00000 G00000] | Standard prefix |
| [http://partsregistry.org/Part:BBa_G00001 G00001] | Standard suffix |
| [http://partsregistry.org/Part:BBa_G00100 G00100] | VF2 primer binding site |
| [http://partsregistry.org/Part:BBa_G00102 G00102] | VR primer binding site |
| [http://partsregistry.org/Part:BBa_I50032 I50032] | p15A replication origin |
| [http://partsregistry.org/Part:BBa_J61107 J61107] | RBS Anderson family |
| [http://partsregistry.org/Part:BBa_J23109 J23100] | Promoter |
| [http://partsregistry.org/Part:BBa_P1004 P1004] | CmR |
| [http://partsregistry.org/Part:BBa_K398400 PhPFD-alpha] | Prefoldin alpha-subunit (Pyrococcus horikoshii OT3) |
| [http://partsregistry.org/Part:BBa_K398401 PhPFD-beta] | Prefoldin beta-subunit (Pyrococcus horikoshii OT3) |
Genbank 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.
