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| - | =Solvent Tolerance=
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| - | 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.
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| - | ==Aim==
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| - | To functionally express the protein prefoldin from ''Pyrococcus horikoshii'' OT3 in ''Escherichia coli'' K12.
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| - | ==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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| - | 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.
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| - | 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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| - | ===Step 1: Formation of Prefoldin BioBrick===
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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.
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| - | === ===
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| - | [[Image:406C.jpg]]
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| - | ===Step 2: Characterization===
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| - | '''Strains:'''
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| - | * Prefoldin: ''E.coli'' K12/407C
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| - | * Negative control: ''E.coli'' K12
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| - | * Positive control: ''Pseudomonas putida OCT''
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| - | 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.
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