Difference between revisions of "Part:BBa K398018"

 
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<partinfo>BBa_K398018 short</partinfo>
 
<partinfo>BBa_K398018 short</partinfo>
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[[Image:TUDelft_Alkane_degradation_route.png|right|410px|thumb|'''Figure 1:'''Complete Alkane degradation pathway, ALDH is the 2nd step herein]]
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ADH, an alcohol dehydrogenase isolated from ''Bacillus thermoleovorans B23'', is capable of converting n-alkanols into the corresponding n-alkanal, the second step in the biodegradation of alkanes.
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===Introduction===
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===Characterization===
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This part was characterized using NAD/NADH enzyme assay. By disrupting the cells in the exponential growth phase (through sonication), adding the substrate dodecanol-1 and analyzing the NADH production using spectrophotometrical analysis (absorbance at 340nm), the activity could be determined. The protein utilizes NAD<sup>+</sup>, thus by determining the NADH production compared to a negative control the activity of the protein can be determined.
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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.
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===Results===
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[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]
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According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to our analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95, which means that the part [https://parts.igem.org/Part:BBa_K398018 BBa_K398018] increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the parts [https://parts.igem.org/Part:BBa_K398005 BBa_K398005] and [https://parts.igem.org/Part:BBa_K398018 BBa_K398018] have catalytic activity; particularly when we used [https://parts.igem.org/Part:BBa_K398018 BBa_K398018] the enzyme activity of ''E. coli'' cell extracts was equivalent to 3% of the ''in vitro'' activity of the positive control (''Pseudomonas putida'').
  
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ADH, an alcohol dehydrogenase isolated from Bacillus thermoleovorans B23, is capable of converting n-alkanols into the corresponding n-alkanal, the second step in the biodegradation of alkanes.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 19:08, 27 October 2010

ADH generator

Figure 1:Complete Alkane degradation pathway, ALDH is the 2nd step herein

ADH, an alcohol dehydrogenase isolated from Bacillus thermoleovorans B23, is capable of converting n-alkanols into the corresponding n-alkanal, the second step in the biodegradation of alkanes.

Introduction

Characterization

This part was characterized using NAD/NADH enzyme assay. By disrupting the cells in the exponential growth phase (through sonication), adding the substrate dodecanol-1 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

Comparison between E. coli ADH activity and our recombinant strain.

According to our results, the E. coli cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to our analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95, which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the parts BBa_K398005 and BBa_K398018 have catalytic activity; particularly when we used BBa_K398018 the enzyme activity of E. coli cell extracts was equivalent to 3% of the in vitro activity of the positive control (Pseudomonas putida).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]