Team:TU Delft/Project/conclusions

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Latest revision as of 23:35, 27 October 2010

Conclusions

We have shown that using the concept of BioBricks it is possible to design an organism that (1) reacts to its environment (sensing), (2) influences the solubility of hydrocarbons (solubility) , (3) exhibits a higher tolerance towards solvents and salts (survival) and (4) implements (parts) of novel pathways (alkane degradation). This chassis could be used for example the biological degradation of residual oil in oil sands tailing waters, or the treatment of waste water from the oil industry.

Sub-projects

Alkane degradation

Assays could be developed for the characterization of the enzymes involved in the first step of alkane degradation (AH-system and LadA). The results show a clear difference in enzyme activity between the strains of interest and the negative control. The strain carrying the AH-system had an enzyme activity of 4.49x10^-2 U/mg dry weight, whereas the negative control was 1.23x10^-3 U/mg dry weight. The strain carrying LadA had an enzyme activity of 3.33x10^-3 U/mg total protein, whereas the negative control was 5.49x10^-4 U/mg total protein.

According to our analysis, the enzymatic activities of our recombinant strain and the negative control are statistically different at confidence level of 0.95, which means that the part BBa_K398018 the alcohol dehydrogenase activity by a factor two. We showed that the parts BBa_K398005 and BBa_K398018 have biological activity; particularly when we used BBa_K398018 the enzyme activity of E. coli cell extracts became equivalent to 3% of the in vitro activity of the positive control (Pseudomonas putida OTR1).

Our results also suggest that the recombinant strains E. coli 029A and E. coli 030A functionally express our biobricks. The biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] 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. These enzymatic activities are equivalent to 33.98% and 42.01% of the Pseudomonas putida aldehyde dehydrogenase activity, respectively. For future work we suggest that more research goes into the fine-tuning of the necessary protein production rates. Furthermore, combining this sub-part with the rest of the sub-projects to create the true chassis would be the next step.

Sensing

After the pre cultures have been grown in glucose, a switch to alkanes as a sole cabon and energy source is required. Therefore we designed a biobrick switch that can sense the absence of glucose: pCaif.

This new promoter combined with B0032 has a GFP production rate of 3.975E07 GFP molecules/second/O.D. during the stationary phase. Moreover, we demonstrated that this promoter is more active the during stationary phase indicating high cAMP levels. Finally, in the presence of a secondary carbon source the GFP production rate decreases again due to the catabolism of the secondary C-source.

Survival

Salt tolerance

Our biobrick has enabled us to increase the salt tolerance of E.coli by an average of 20%. The presented BioBrick probably reduces one of several effects caused by salt stress. A complete tolerance using a single protein seems unrealistic, a combination of different could further increase the tolerance to salt.

Solvent tolerance

Our BioBrick has enabled us to increase the solvent tolerance of E.coli when n-hexane is present in the culture medium at high concentrations. According to our findings, the growth rate of E.coli is improved 60% at a n-hexane concentration of 10%(v/v).

Solubility

To overcome the mass‐transfer limitation between the water and oil phase, a gene encoding for AlnA, a protein with emulsifying properties was expressed. The increased solubility is about 20%. The solubility was determined by a new method. We suggest that in future research the protein is tagged, so it can be isolated with higher purity.

RBS Characterization

In order to fine-tune the protein expressions of the alkane degrading genes we've characterized 5 members of the Anderson RBS family using an improved protein production model taking dilution and protein degradation into account. The following relative efficiencies were found:

RBS	Efficiency
[http://partsregistry.org/Part:BBa_J61100 J61100]	1.20%
[http://partsregistry.org/Part:BBa_J61101 J61101]	11.9%
[http://partsregistry.org/Part:BBa_J61107 J61107]	7.70%
[http://partsregistry.org/Part:BBa_J61117 J61117]	1.26%
[http://partsregistry.org/Part:BBa_J61127 J61127]	6.52%
[http://partsregistry.org/Part:BBa_B0032 B0032]	30.0%

@@ Line 16: / Line 16: @@
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-According to our analysis, the enzymatic activities of our recombinant strain and the negative control 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 biological activity; particularly when we used BBa_K398018 the enzyme activity of E. coli cell extracts became equivalent to 3% of the in vitro activity of the positive control: ''Pseudomonas putida'' a natural alkane-degrading bacterium.
+Assays could be developed for the characterization of the enzymes involved in the first step of alkane degradation (AH-system and LadA). The results show a clear difference in enzyme activity between the strains of interest and the negative control. The strain carrying the AH-system had an enzyme activity of 4.49x10<sup>-2</sup> U/mg dry weight, whereas the negative control was 1.23x10<sup>-3</sup> U/mg dry weight. The strain carrying LadA had an enzyme activity of 3.33x10<sup>-3</sup> U/mg total protein, whereas the negative control was 5.49x10<sup>-4</sup> U/mg total protein.
+According to our analysis, the enzymatic activities of our recombinant strain and the negative control are statistically different at confidence level of 0.95, which means that the part BBa_K398018 the alcohol dehydrogenase activity by a factor two. We showed that the parts BBa_K398005 and BBa_K398018 have biological activity; particularly when we used BBa_K398018 the enzyme activity of E. coli cell extracts became equivalent to 3% of the in vitro activity of the positive control (''Pseudomonas putida'' OTR1).
+Our results also suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] 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. These enzymatic activities are equivalent to 33.98% and 42.01% of the Pseudomonas putida aldehyde dehydrogenase activity, respectively. For future work we suggest that more research goes into the fine-tuning of the necessary protein production rates. Furthermore, combining this sub-part with the rest of the sub-projects to create the true chassis would be the next step.
-Our results also suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. From our data we conclude that the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] 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.
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@@ Line 32: / Line 35: @@
 Sensing
 </a></h3></html>
-After the pre cultures are grown in glucose, it needs to switch to alkanes as a feedstock. Therefore we designed a biobrick switch that can sense the absence of glucose: pCaif.
+After the pre cultures have been grown in glucose, a switch to alkanes as a sole cabon and energy source is required. Therefore we designed a biobrick switch that can sense the absence of glucose: pCaif.
+This new promoter combined with B0032 has a GFP production rate of 3.975E07 GFP molecules/second/O.D. during the stationary phase. Moreover, we demonstrated that this promoter is more active the during stationary phase indicating high cAMP levels. Finally, in the presence of a secondary carbon source the GFP production rate decreases again due to the catabolism of the secondary C-source.
-This new promoter combined with B0032 has a GFP production rate of 3.975E07 GFP molecules/second/O.D. during the stationary phase. Moreover, we demonstrated that this promoter is more active during stationary phase (high cAMP levels). Finally, in the presence of a secondary carbon source the GFP production rate decreases due to the catabolism of the secondary C-source.
 <html></div><br style="clear:both;" /></html>
@@ Line 46: / Line 51: @@
 ====Salt tolerance====
-Our biobrick has enabled us to increase the salt tolerance of E.coli by an average of 20%. But due to the range of effects caused by increased salt stress, complete tolerance using a single protein is impossible. As such we hope to have made a first step and that the future iGEM teams will be able to build upon this knowledge.
+Our biobrick has enabled us to increase the salt tolerance of E.coli by an average of 20%. The presented BioBrick probably reduces one of several effects caused by salt stress. A complete tolerance using a single protein seems unrealistic, a combination of different could further increase the tolerance to salt.
 ====Solvent tolerance====
-Our biobrick has enabled us to increase the solvent tolerance of ''E.coli'' when n-hexane is present in the culture medium at high concentrations. According to our findings, the growth rate of ''E.coli'' is improved 60% at a n-hexane concentration of 10%(v/v).
+Our BioBrick has enabled us to increase the solvent tolerance of ''E.coli'' when n-hexane is present in the culture medium at high concentrations. According to our findings, the growth rate of ''E.coli'' is improved 60% at a n-hexane concentration of 10%(v/v).
 <html></div><br style="clear:both;" /></html>
@@ Line 60: / Line 65: @@
 Solubility
 </a></h3></html>
-To overcome the mass‐transfer limitation between the water and oil fase, a gene encoding for [[Team:TU_Delft/Project/solubility/alna|AlnA]], a protein with emulsifying properties was expressed. The [[Team:TU_Delft/Project/solubility/results|increased solubility]] of about 20% was determined by [[Team:TU_Delft/Project/solubility/characterization|a new method]]. We suggest that in future research the protein is tagged, so it can be isolated with higher purity.
+To overcome the mass‐transfer limitation between the water and oil phase, a gene encoding for [[Team:TU_Delft/Project/solubility/parts|AlnA]], a protein with emulsifying properties was expressed. The [[Team:TU_Delft/Project/solubility/results|increased solubility]] is about 20%. The solubility was determined by [[Team:TU_Delft/Project/solubility/characterization|a new method]]. We suggest that in future research the protein is tagged, so it can be isolated with higher purity.
 <html><br /><br /></div><br style="clear:both;" /></html>
@@ Line 72: / Line 77: @@
 </a></h3></html>
-In order to tune the protein expressions of the alkane degrading genes we've [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization characterized] 5 members of the Anderson RBS family using an improved protein production model taking dilution into account. The following relative efficiencies were found:
+In order to fine-tune the protein expressions of the alkane degrading genes we've [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization characterized] 5 members of the Anderson RBS family using an improved protein production model taking dilution and protein degradation into account. The following relative efficiencies were found:
 {|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
 |<b>RBS</b>