Team:TU Delft/project/hydrocarbon tolerance

From 2010.igem.org

(Difference between revisions)
(→Proposed Method)
(→Proposed Method)
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==Proposed Method==
==Proposed Method==
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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)'''
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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)'''
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For this part of our project, we will insert and functionally express the ''alpha'' and ''beta'' sub-units of ''Pyrococcus horikoshii'' prefoldin; this protein confers solvent tolerance to ''E. coli'' K12 cells. 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.
+
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.  
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.  
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'''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.
'''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.
=== ===
=== ===
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[[Image:406C.jpg]]
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[[Image:406C.jpg|650px|left]]  
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 +
|'''Feature'''
 +
|'''Function'''
 +
|-
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|[http://partsregistry.org/Part:BBa_K398001 alkB2]
 +
|Alkane 1-monooxygenase (Gordonia sp. TF6)
 +
|-
 +
|[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]
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|Standard prefix
 +
|-
 +
|[http://partsregistry.org/Part:BBa_G00001 G00001]
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|Standard suffix
 +
|-
 +
|[http://partsregistry.org/Part:BBa_G00100 G00100]
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|VF2 primer binding site
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|-
 +
|[http://partsregistry.org/Part:BBa_G00102 G00102]
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|VR primer binding site
 +
|-
 +
|[http://partsregistry.org/Part:BBa_I50032 I50032]
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|p15A replication origin
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|-
 +
|[http://partsregistry.org/Part:BBa_J61100 J61100]
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|RBS Anderson family
 +
|-
 +
|[http://partsregistry.org/Part:BBa_J23109 J23100]
 +
|Promoter
 +
|-
 +
|[http://partsregistry.org/Part:BBa_P1005 P1005]
 +
|TetR
 +
|-
 +
|[http://partsregistry.org/Part:BBa_K398002 rubA3]
 +
|Rubredoxin A3 (Gordonia sp. TF6)
 +
|-
 +
|[http://partsregistry.org/Part:BBa_K398003 rubA4]
 +
|Rubredoxin A4 (Gordonia sp. TF6)
 +
|-
 +
|[http://partsregistry.org/Part:BBa_K398004 rubR]
 +
|Rubredoxin reductase (Gordonia sp. TF6)
 +
|-
 +
|}

Revision as of 09:08, 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)


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.

Retrieved from "http://2010.igem.org/Team:TU_Delft/project/hydrocarbon_tolerance"