Difference between revisions of "Part:BBa K398030"
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ALDH is an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids (see figure 1), which can then be further degraded through β-oxidation. Implementation of this BioBrick into ''E.coli'' resulted in a 42% increased enzyme activity compared to the negative control. | ALDH is an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids (see figure 1), which can then be further degraded through β-oxidation. Implementation of this BioBrick into ''E.coli'' resulted in a 42% increased enzyme activity compared to the negative control. | ||
| − | '''NOTE''' ''This part contains the same protein as [https://parts.igem.org/Part:BBa_K398029 BBa_K398029] but with a stronger promoter-RBS region. The following text was copied from the [https://parts.igem.org/Part:BBa_K398029 BBa_K398029 parts page].'' | + | '''NOTE''' ''This part contains the same protein as [https://parts.igem.org/Part:BBa_K398029 BBa_K398029] but with a stronger promoter-RBS region. Figure 3 shows both activities. The following text was copied from the [https://parts.igem.org/Part:BBa_K398029 BBa_K398029 parts page].'' |
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===Introduction=== | ===Introduction=== | ||
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===Results=== | ===Results=== | ||
| − | [[Image:TUDelftALDH_map.jpg|410px|thumb|right|Comparison of ALDH activities in the different cell extracts tested in our study.]] | + | [[Image:TUDelftALDH_map.jpg|410px|thumb|right|'''Figure 2:''' Comparison of ALDH activities in the different cell extracts tested in our study.]] |
[http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results TU Delft 2010 results] suggest that the cell extracts obtained from the recombinant strain ''E. coli'' 029A, which expresses this part, have a dodecanal dehydrogenase activity twice as high as the control strain (''E. coli'' with the empty plasmid pSB1A2). This activity is equivalent to 33.98% of the cell extract of ''Pseudomonas putida'' growing on octane as sole carbon source. | [http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results TU Delft 2010 results] suggest that the cell extracts obtained from the recombinant strain ''E. coli'' 029A, which expresses this part, have a dodecanal dehydrogenase activity twice as high as the control strain (''E. coli'' with the empty plasmid pSB1A2). This activity is equivalent to 33.98% of the cell extract of ''Pseudomonas putida'' growing on octane as sole carbon source. | ||
| − | [[Image:TUDelftALDH_final.jpg|470px|thumb|left|Comparison of ALDH activities in the different strains tested in this study]] | + | |
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| + | [[Image:TUDelftALDH_final.jpg|470px|thumb|left|'''Figure 3:''' Comparison of ALDH activities in the different strains tested in this study]] | ||
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===References=== | ===References=== | ||
#'''Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa.''' Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans B23''. ''Extremophiles'' 14:33-39 ('''2010''') | #'''Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa.''' Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans B23''. ''Extremophiles'' 14:33-39 ('''2010''') | ||
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Revision as of 21:00, 27 October 2010
ALDH generator (over-expression)
ALDH is an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids (see figure 1), which can then be further degraded through β-oxidation. Implementation of this BioBrick into E.coli resulted in a 42% increased enzyme activity compared to the negative control.
NOTE This part contains the same protein as BBa_K398029 but with a stronger promoter-RBS region. Figure 3 shows both activities. The following text was copied from the BBa_K398029 parts page.
Introduction
This part consists of an aldehyde dehydrogenase from the thermophile Geobacillus thermoleovorans B23. It functions as an octamer, requiring NAD+ as coenzyme. The optimum condition for activity lies at temperatures between 50 and 55 degrees celsius and and a pH of 10. You can see our original plasmid map below.
Characterization
This part was characterized using NAD/NADH enzyme assay. By disrupting the cells in the exponential growth phase (through sonication), adding the substrate dodecanal and analyzing the NADH production using spectrophotometrical analysis (absorbance at 340nm), the activity could be determined. The protein utilizes NAD+, thus by determining the NADH production compared to a negative control the activity of the protein can be determined.
The [http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation characterization of BBa_K398029] has been described on the TU Delft iGEM Team 2010 wiki.
Results
[http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results TU Delft 2010 results] suggest that the cell extracts obtained from the recombinant strain E. coli 029A, which expresses this part, have a dodecanal dehydrogenase activity twice as high as the control strain (E. coli with the empty plasmid pSB1A2). This activity is equivalent to 33.98% of the cell extract of Pseudomonas putida growing on octane as sole carbon source.
References
- Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa. Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23. Extremophiles 14:33-39 (2010)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]