Team:TU Delft/Project/conclusions

From 2010.igem.org

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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 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.
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CONCLUSION
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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.
===Anderson RBS Characterizations===
===Anderson RBS Characterizations===

Revision as of 12:36, 27 October 2010

Conclusions

Our goal was to tackle the biological conversion of hydrocarbons in an aqueous environment. The basis of the project was generating a "biological chassis", which provides the framework for varying and multiple characteristics needed for the conversion of hydrocarbons, including considerations like conversion ability, hydrocarbon tolerance/solubility and halo (salt) tolerance. This chassis could than be used in for example specifically the biological degradation of oil particles in oil sands tailing water.

Alkane Degradation

Survival

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.

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 fase, a gene encoding for AlnA, a protein with emulsifying properties was expressed. The increased solubility of about 20% was determined by a new method. We suggest that in future research the protein is tagged, so it can be isolated with higher purity.

Sensing

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.

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.

Anderson RBS Characterizations

In order to 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 into account. The following relative efficiencies were found:

RBS Efficiency
J61100 1.20%
J61101 11.9%
J61107 7.70%
J61117 1.26%
J61127 6.52%
B0032 30.0%