Team:TU Delft/Project/alkane-degradation/results

From 2010.igem.org

(Difference between revisions)
(→Characterization of the alkane hydroxylase system)
(→Characterization of the long-chain alkane monooxygenase; LadA)
Line 50: Line 50:
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.
 +
 +
 +
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
Line 94: Line 97:
|3.33E-03
|3.33E-03
|}
|}
-
 
-
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]
 
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===

Revision as of 19:21, 27 October 2010

CharacterizationResultsParts

Alkane Degradation Results & Conclusions

Figure 1 – Schematic description of the alkane degradation pathway with the corresponding genes.

Characterization of the alkane hydroxylase system

The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. From the ratios we may conclude that the samples obtained from the E.coli strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. For more information about our findings CLICK HERE.

Strain Ratio octane/undecane Standard deviation [%] Octane converted [umol] Protein [mg] Enzymatic activity [U/mg protein]
Blank 0.832 6.16 0.00 0.00 0.00
E.coli K12 negative control 0.821 13.3 1.34 1.51 1.23E-3
E.coli K12 strain #1 (AH-system) 0.652 14.4 21.6 1.70 1.77E-2
E.coli K12 strain #2 (AH-system) 0.439 3.96 47.0 1.46 4.49E-2

Characterization of the long-chain alkane monooxygenase; LadA

The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.


We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, CLICK ON THIS LINK

<b>Strain Average ratio hexadecane/undecane Standard deviation [%] Hexadecane converted [umol] Protein [mg] Enzymatic activity [U/mg protein]
Blank 0.900 3.19 0.00 0.00 0.00
E.coli K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002]) 0.807 3.48 1.76 4.46 5.49E-04
E.coli K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017]) 0.600 15.4 5.68 4.58 1.72E-03
E.coli K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017]) 0.532 4.25 6.95 3.18 3.03E-03
E.coli TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027]) 0.492 2.98 7.71 3.22 3.33E-03

Characterization of the Alcohol DeHydrogenase (ADH) system

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 expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control Pseudomonas putida.

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

If you are interested in knowing more about our findings, CLICK ON THIS LINK.

Characterization of the ALdehyde DeHydrogenase system

USEFUL LITERATURE AND REFERENCES

  1. Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23" Extremophiles (2010) 14:33-39.
  2. http://mbel.kaist.ac.kr/lab/research/protein_en1.html
  3. Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in Escherichia coli" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.

CharacterizationResultsParts

Retrieved from "http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results"