"
Team:TU Delft/Project/alkane-degradation/characterization
From 2010.igem.org
(→Alkane Degradation Characterization) |
(→Characterization of the Alkane Degradation Parts) |
||
| Line 5: | Line 5: | ||
===Characterization of the Alkane Degradation Parts=== | ===Characterization of the Alkane Degradation Parts=== | ||
| - | + | In summary, the following strains were characterized: | |
| - | *''E.coli'' K12 | + | *''E.coli'' K12 carrying BBa_K398014 in pSB1A2 (AH system) |
| - | *''E.coli'' K12 | + | *''E.coli'' K12 carrying BBa_K398017 in pSB1A2 (LadA) |
| - | *''E.coli'' | + | *''E.coli'' TOP10 carrying BBa_K398027 in pSB1A2 (LadA) |
| - | *''E.coli'' K12 | + | *''E.coli'' K12 carrying BBa_K398018 in pSB1A2 (ADH) |
| - | *''E.coli'' K12 | + | *''E.coli'' K12 carrying BBa_K398029 in pSB1A2 (ALDH) |
| - | The characterization will | + | The characterization will generally be executed along with an 'empty' plasmid carrying strain: |
| - | + | *''E.coli'' K12 carrying BBa_J13002 in pSB1A2 ('empty' plasmid) | |
| - | + | ||
| - | + | ||
===Characterization of growth=== | ===Characterization of growth=== | ||
Revision as of 12:09, 24 October 2010
Alkane Degradation Characterization
Characterization of the Alkane Degradation Parts
In summary, the following strains were characterized:
- E.coli K12 carrying BBa_K398014 in pSB1A2 (AH system)
- E.coli K12 carrying BBa_K398017 in pSB1A2 (LadA)
- E.coli TOP10 carrying BBa_K398027 in pSB1A2 (LadA)
- E.coli K12 carrying BBa_K398018 in pSB1A2 (ADH)
- E.coli K12 carrying BBa_K398029 in pSB1A2 (ALDH)
The characterization will generally be executed along with an 'empty' plasmid carrying strain:
- E.coli K12 carrying BBa_J13002 in pSB1A2 ('empty' plasmid)
Characterization of growth
The preliminary characterization will aim to determine the presence of growth on any one of the following alkanes:
- octane (C8)
- undecane (C11)
- dodecane (C12)
- tetradecane (C14)
- heptadecane (C17)
- octadecane (C18)
For the growth characterization on short-chain and long-chain alkanes an E.coli K12 strain containing BBa_K398015 and BBa_K398022 (or intermediates thereof) is inoculated into M9 minimal medium containing 5% v/v ratio of the respective alkane on a 96-well plate with 200 μL volume per well. Growth will be determined o/n by absorbance at 600nm with intervals of 10 minutes.
Characterization of enzyme functionality
Parallel to this, resting-cell assays will be performed on growth-inhibited E.coli K12 strains containing the constructs described earlier. These assays will indicate the presence or absence of the desired enzymes, regardless of the alkane’s utilization for growth. The hydrocarbon compositions will be determined by gas chromatography analysis. For the intracellular enzymes, the cells were lysed and treated with the appropriate akanols and alkanals.
