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

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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.

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

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.

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.

<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

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

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

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.
http://mbel.kaist.ac.kr/lab/research/protein_en1.html
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.

@@ Line 99: / Line 99: @@
 ===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH 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 018A 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''.
+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''.
 [[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]
+If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].
 ===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===