From 2010.igem.org
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| | ===Characterization of the alkane hydroxylase system=== | | ===Characterization of the alkane hydroxylase system=== |
| - | ====Growth analysis====
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| - | We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.
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| - | ====Resting-cell assays====
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| - | The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].
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| - | The following are examples of typical chromatographs obtained:
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| - | <html><span style="display:block;width:100%;clear:both;"> </span></html>
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| - | [[Image:Blank_C-8.png|400px|thumb|left|Chromatograph of blank containing octane.]]
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| - | [[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]
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| - | [[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]
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| - | <html><span style="display:block;width:100%;clear:both;"> </span></html>
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| - | The peaks shown in the chromatographs belong to the following compounds:
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| - | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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| - | |<b>Retention time [min]</b>
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| - | |<b>Compound</b>
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| - | |-
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| - | |~2.2
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| - | |Ethyl acetate (solvent)
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| - | |-
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| - | |~5.2
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| - | |Octane (substrate)
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| - | |-
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| - | |~11.8
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| - | |Undecane (internal standard, 0.1% v/v of solvent)
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| - | |}
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| - | The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.
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| - | 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.
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| - | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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| - | |<b>Strain</b>
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| - | |<b>Ratio octane/undecane</b>
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| - | |<b>Standard deviation [%]</b>
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| - | |<b>Octane converted [umol]</b>
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| - | |<b>Protein [mg]</b>
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| - | |<b>Enzymatic activity [U/mg protein]
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| - | |-
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| - | |Blank
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| - | |0.832
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| - | |6.16
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| - | |0.00
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| - | |0.00
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| - | |0.00
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| - | |-
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| - | |''E.coli'' K12 negative control
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| - | |0.821
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| - | |13.3
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| - | |1.34
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| - | |1.51
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| - | |1.23E-3
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| - | |-
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| - | |''E.coli'' K12 strain #1 (AH-system)
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| - | |0.652
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| - | |14.4
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| - | |21.6
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| - | |1.70
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| - | |1.77E-2
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| - | |-
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| - | |''E.coli'' K12 strain #2 (AH-system)
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| - | |0.439
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| - | |3.96
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| - | |47.0
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| - | |1.46
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| - | |4.49E-2
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| - | |}
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| - | 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. Using a two-point calibration method, where we set the ratio of 0.898 equal to the total amount of octane added (16.2 uL, 99.7 umol), we can estimate the enzymatic activity of the AH-system. We know the reaction time (12 h.) and the mg of protein for each sample (using the conversion factor 0.41g/L/OD). See the table below for details.
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| - | ====Conclusions====
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| - | We've succeeded in characterizing the activity of the alkane hydroxylase system using gas chromatography. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system.
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| - | The lack of an octanol peak in the GC-graphs above could be caused by the octanol being trapped by the cell, and thus not showing up in the organic (EtOAc) phase. Another likely explanation is that the octanol is readily converted by an aspecific alcohol dehydrogenase native to the ''E.coli'' K12 strain.
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| - | In the future we would like to perform a resting-cell assay using ''P. putida'' and determine the enzymatic activity needed to support growth. A comparison of this natural-degrader value with the biological activity we've found of 0.045 U/mg would give more information on the growth feasibility of a strain carring the AH-system BioBrick on octane.
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| - | Furthermore, a study could be done on the AH-system over time to find out more information on the kinetics of the system.
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| | ===Characterization of the long-chain alkane monooxygenase; LadA=== | | ===Characterization of the long-chain alkane monooxygenase; LadA=== |
Revision as of 19:01, 27 October 2010
Alkane Degradation Results & Conclusions
Figure 1 – Schematic description of the alkane degradation pathway with the corresponding genes.
Characterization of the alkane hydroxylase system
Characterization of the long-chain alkane monooxygenase; LadA
Enzyme activity assay based on NADH absorbance
Curves of the NADH absorbance for the blank, negative control and strains of interest displayed no significant differences, thus this method was abandoned. The reason for this could be explained by a lack of cofactors, a substrate diffusion limitation or even substrate inhibition.
Enzyme activity assay based on GC-analysis
Our enzyme activity protocol for LadA was loosely based an article by Maeng et al,1996. Homogenization of the alkane in the 50mM Tris buffer was achieved by adding Triton-X100, boiling and sonication. After 12 hours the residual alkane was extracted using EtOAc.
The following are typical examples of the chromatographs obtained:
Chromatograph of blank containing hexadecane
Chromatograph of the negative control for the enzyme kinetics of the alkane monooxygenase. The negative control consisted of
E.coli K12 carrying [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] in pSB1A2.
Chromatograph of sample obtained from
E.coli K12 carrying [http://partsregistry.org/Part:BBa_K398017 BBa_K398017] in pSB1A2.
Chromatograph of sample obtained from
E.coli TOP10 carrying [http://partsregistry.org/Part:BBa_K398027 BBa_K398027] in pSB1A2.
The peaks shown in the chromatographs belong to the following compounds:
| Retention time [min]
| Compound
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| ~2.2
| Ethyl acetate (solvent)
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| ~11.8
| Undecane (internal standard, 0.1% v/v of solvent)
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| ~20.0
| Hexadecane (substrate)
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The analysis of the GC graphs was analogous to that of the AH system, described above. We made use of the internal standards present in each sample to determine the ratio of the surface areas of hexadecane vs. internal standard. These ratios can be related to the amounts of hexadecane added to eventually 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.
| Strain
| Average ratio hexadecane/undecane
| Standard deviation [%]
| Hexadecane converted [umol]
| Protein [mg]
| Enzymatic activity [U/mg protein]
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| Blank
| 0.900
| 3.19
| 0.00
| 0.00
| 0.00
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| E.coli K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])
| 0.807
| 3.48
| 1.76
| 4.46
| 5.49E-04
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| E.coli K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])
| 0.600
| 15.4
| 5.68
| 4.58
| 1.72E-03
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| E.coli K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])
| 0.532
| 4.25
| 6.95
| 3.18
| 3.03E-03
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| E.coli TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])
| 0.492
| 2.98
| 7.71
| 3.22
| 3.33E-03
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Conclusions
LadA was characterized succesfully using an enzyme kinetics assay coupled to gas chromatography. We see a significant increase in enzyme activity in the strains carrying the ladA protein generator BioBrick compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein compared to 0.55E-03 of the negative control strain.
Further studies into the enzymatic activity needed to sustain growth could be performed in future. This will give us information on the implementation of this BioBrick into our complete alkane degradation chassis.
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.
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