Team:TU Delft/Project/conclusions

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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%