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

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==Alkane Degradation Results & Conclusions==
==Alkane Degradation Results & Conclusions==
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[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]
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[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1.''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]
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===Characterization of the alkane hydroxylase system===
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===[http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system (AlkB2-system)]===
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====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 [http://2010.igem.org/Team:TU_Delft#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 [http://2010.igem.org/Team:TU_Delft#Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [http://2010.igem.org/Team:TU_Delft#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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From the ratios hexadecane/undecane obtained from GC chromatograms, 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.  
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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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For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.
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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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[[Image:TUDelft_AlkB2_Total.png|600px|thumb|center|'''Figure 2.''' Enzyme activity [U/mg total protein] of alkane hydroxylase system as compared to the negative control ''E.coli'' K12 strain]]
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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. If we assume that a negligible amount hydrocarbon will remain present in the aqueous phase, we can state that 2.5 uL (
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===[http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase (LadA)]===
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===LadA===
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The analysis of the obtained GC graphs allowed us to estimate the enzymatic activity. 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, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.
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===Alcohol DeHydrogenase (ADH) system===
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[[Image:TUDelft_LadA Total.png|600px|thumb|center|'''Figure 3.''' Enzyme activity [U/mg lysate] of the alkane monooxygenase LadA as compared to the negative control ''E.coli'' K12 strain]]
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==== Results ===
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=====Growth on alcohols as sole carbon source=====
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*As we described before, we tried to grow our strain ''Escherichia coli'' 018A (Biobrick BBa_K398018 on the plasmid pSB1A2) on the alkanols Octanol-1 and Dodecanol-1. No growth was observed after 48 hours. We abandoned this experiment after this result.
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=====Resting cell assays=====
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*We grew ''Escherichia coli'' 018A and ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.
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*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [http://2010.igem.org/Team:TU_Delft#Resting-cell_assays_for_E.coli standard protocol]
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{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1";
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===[http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of Alcohol Dehydrogenase (ADH)]===
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|'''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 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''.
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|'''O.D.'''
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|-
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|J13002
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|0.327
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|-
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|018A
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|0.338
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|}
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*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanals or alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:
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[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|'''Figure 1''' – Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]
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If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.
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[[Image:TUDelftADH_final.jpg|600px|thumb|center|'''Figure 4.''' Comparison between E. coli ADH activity and our recombinant strain. ]]
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*According to our results there was no degradation of octanol-1 ='( , after this experiment the protocol was abandoned. We think that the cells lack of transporters for long-chain alcohols, we preferred to check the cell extract in order to know if there was any biological activity in the cytoplasma.
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===[http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of Aldehyde Dehydrogenase (ALDH)]===
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=====NADH production in cell extracts=====
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Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold.  
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*Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.
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{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"
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|Strain
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|O.D. 600nm
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|-
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|J13002 (1)
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|0.654
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|-
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|J13002 (2)
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|0.621
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|-
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|018A (1)
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|0.615
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|-
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|018A (2)
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|0.580
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|}
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*Cytoplasmic proteins were extracted using our standard [http://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a  standard curve for protein quantification was prepared.
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[[Image:TUDelftADH_bradford.jpg|600px|thumb|center|'''Figure 1''' – Standard curve for protein determination.]]
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*The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.
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{| style="color:black; background-color:white;" cellpadding="4" cellspacing="0" border="1"
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Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively.  
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|Sample
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|'''O.D. 562 nm'''
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|'''Total ug of protein'''
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|'''ug/uL'''
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|-
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|J13002(1A)
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|0.135
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|10.393
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|1.0393
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|-
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|J13002 (1B)
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|0.135
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|10.393
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|1.0393
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|-
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|J13002 (2A)
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|0.128
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|9.7363
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|0.9736
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|-
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|J13002 (2B)
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|0.129
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|9.8302
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|0.9830
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|-
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|018A (1A)
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|0.097
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|6.8275
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|0.6827
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|-
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|018A (1B)
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|0.091
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|6.2645
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|0.6264
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|-
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|018A (2A)
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|0.092
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|6.3583
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|0.6358
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|-
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|018A (2B)
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|0.092
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|6.3583
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|0.6358
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|}
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*The alcohol dehydrogenase activity was measured using the standard [http://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [[ Image:TUDelft_ADH_raw.xls]]
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If you want to know more about of our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ALDH|detailed ALDH results]] page.  
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*After the data treatment, the results that we obtained are the following:
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It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.
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[[Image:TUDelftADH_pH95.jpg|400px]]
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[[Image:TUDelftALDH_final.jpg|600px|thumb|center|'''Figure 5.''' Comparison of ALDH activities in the different strains tested in this study]]
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[[Image:TUDelftADH_pH8.jpg|400px]]
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*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. 
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===References===
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#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.
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Maybe you are not familiar with the term "katal", officially the katal is the STANDARD UNIT FOR CATALYTIC ACTIVITY in the International System of Units; the katal is defined as the amount of enzyme that converts 1 mol of substrate each second. [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] to know more about it, and because people are not that familiar with the term, we report the activities in U/mg. One enzyme activity unit is defined as the amount of protein that converts 1 umol of substrate each minute.
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#http://mbel.kaist.ac.kr/lab/research/protein_en1.html
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#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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<html>
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<table>
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<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr>
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<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr>
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<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr>
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<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr>
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<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr>
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<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr>
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<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr>
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<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr>
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</table>
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</html>
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==== Conclusions ====
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This shows two things.
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That E.Coli is partly able to degrade Dodecanol on it's own. And that this activity is greatly increased (43.83% normalized for the amount of protein) with the addition of the ADH gene.
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It is also clear that eventhough ADH is from a thermophilic organism it loses most (63.5%) of it's activity after heating. the raw data for these calculations can be seen below.
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(Although this decrease in activity could also be attributed to the host organism lacking the proper post translational modifications.)
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===ALDH===
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==== Growth on alcohols as sole carbon source====
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As in the case of alkane monooxygenases, our first attempt was to grow our recombinant strains on long-chain alcohols and aldehydes; hoping that E. coli will do the further degradation steps. For that purpose, we used octanol-1 and dodecanol-1 for the ADH system; whereas we used octanal and dodecanal for the ALDH system.  If everything goes well, we should see microbial growth on minimal medium with these compounds as sole carbon source.
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==== Resting cell assays====
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Our second attempt was to grow cells, until the exponential phase is reached; then the cells were collected and the spent medium was changed for a buffer with glucose that lacked of N-source, this was done in order to avoid more biomass production. Additionally we added alcohols and aldehydes to the mixture, so that if our part is working they will be converted in other molecules: alcohols to aldehydes and aldehydes to alkanoic acids.
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With this approach, we are hoping that the enzymes produced during the growth phase will remain inside the cells. The glucose added will serve for maintenance purposes and co-factor regeneration. If everything works well, there will be some nice peaks appearing on our GC analysis.
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==== NADH production in cell extracts====
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We called this assay: THE LAST RESOURCE  =s
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Basically, we grew cells until an O.D. (600nm) between 0.5-1.0. We needed chubby healthy happy exponential-growth cells because most of our constructions are meant for protein production in this growth phase.
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Once, we got a lot of cells... we disrupted them by sonication and we analyze the NADH production by their guts using a simple spectrophotometrical analysis. If our parts are working, we should see a higher NADH production when the substrate (dodecanol-1 or dodecanal) to the buffer with microbial guts and NAD buffer. We can use this method  because NAD is the natural co-factor of our proteins. Fortunately for us, NAD reduction could be easily quantified by absorbance measurements at 340 nm.
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We called this protocol "the last resource" because the others didn't work. Which means that alcohols and aldehydes do not cross the cell membrane, thus in order to grow cell on these compounds as sole carbon sources... WE NEEDED TO ADD TRANSPORTERS =(
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====BBa_K398030 and BBa_K398029====
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Both Biobricks are the protein generators of Bt-ALDH. We compared the activities of bot parts, parental strain with an empty plasmid (J13002) and a natural oil-degrader (Pseudomonas putida).
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[[Image:TU_Delft_ALDH_comp.jpg]]
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398029 BBa_K398029 in the '''parts registry''']
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'''Specified Components'''
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<partinfo>K398029 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398029 Designer</partinfo>
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'''Status:''' <partinfo>K398029 Status</partinfo>
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398030 BBa_K398030 in the '''parts registry''']
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'''Specified Components'''
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<partinfo>K398206 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398030 Designer</partinfo>
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'''Status:''' <partinfo>K398030 Status</partinfo>
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html>
<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html>

Latest revision as of 22:32, 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 (AlkB2-system)

From the ratios hexadecane/undecane obtained from GC chromatograms, 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, read the detailed alkane hydroxylase results page.

Figure 2. Enzyme activity [U/mg total protein] of alkane hydroxylase system as compared to the negative control E.coli K12 strain

Characterization of the long-chain alkane monooxygenase (LadA)

The analysis of the obtained GC graphs allowed us to estimate the enzymatic activity. 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, read the detailed LadA results page.

Figure 3. Enzyme activity [U/mg lysate] of the alkane monooxygenase LadA as compared to the negative control E.coli K12 strain

Characterization of Alcohol Dehydrogenase (ADH)

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 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.

If you are interested in knowing more about our findings, read the detailed ADH results page.

Figure 4. Comparison between E. coli ADH activity and our recombinant strain.

Characterization of Aldehyde Dehydrogenase (ALDH)

Our results suggest that the recombinant strains E. coli 029A and E. coli 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination BBa_J23100-BBa_J61117 increases the dodecanal dehydrogenase activity in E. coli cell extracts 2-fold; whereas the expression of the same protein using the part BBa_J13002 as promoter-rbs combo increases the same activity 3-fold.

Moreover, the enzymatic activities measured for the constructs BBa_K398029 and BBa_K398030 were equivalent to 33.98% and 42.01% of the Pseudomonas putida aldehyde dehydrogenase activity, respectively.

If you want to know more about of our findings, read the detailed ALDH results page.

It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.

Figure 5. Comparison of ALDH activities in the different strains tested in this study


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.

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