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From 2010.igem.org

Contents 1 Synthetic biology 2 The problem (oil in water) 3 Our solution (strategy) 3.1 Part I – Alkane degradation 3.2 Part II – Tolerance 3.3 Part III - Solubility 3.4 Part IV – Genetic regulation

Synthetic biology

Introduction to synthetic biology

The problem (oil in water)

Introduction to the problem

Our solution (strategy)

In order to solve the problem of oil in water, as seen in the Oil Sands tailing waters, the iGEM TU Delft 2010 team has designed a strategy composed of various parts. Each part focuses on a certain challenge faced when removing hydrocarbons from aqueous environments. Together, these subparts form a complete chassis for a hydrocarbon-degrading microorganism. Our general strategy is to create BioBricks® that will facilitate each sub-part of our project within E.coli. This will be done by taking specific genes from various organisms from nature and placing them in a BioBrick®, after which there functioning can be evaluated within E.coli.

Part I – Alkane degradation

First and foremost the organism will have to be able to degrade oil. Oil consists of various hydrocarbon types and sizes, and for this challenge we decided to start with alkanes. There are a number of microorganisms that are known to degrade alkanes, and of these, genes from three different organisms will be used for the conversion of short chain alkanes, long chain alkanes and long chain alcohols and aldehydes, the latter two being intermediates of the long chain alkane degradation pathway. For the further degradation we will rely on the in-house genes of E.coli such as the β-oxidation pathway.

Part II – Tolerance

Hydrocarbons and salts in too high concentrations are known to be toxic to cells. It was found that organisms that are naturally hydrocarbon tolerant produce chaperones that maintain the cellular activity. There are also numerous organisms that can live in salty environments. By using genes from these organisms we hope to create BioBricks® that will facilitate salt tolerance as well as hydrocarbon tolerance.

Part III - Solubility

Oil and water don’t mix; the low solubility of hydrocarbons in water very likely could form a challenge for a hydrocarbon-degrading organism. To overcome this challenge a BioBrick® will be made containing genes encoding for an emulsifier that will increase the solubility.

Part IV – Genetic regulation

In order to have efficient cell growth, it is important to develop a system that activates gene expression at the optimal moment in time. An alkane sensing mechanisms described in literature will be adapted and incorporated using the BioBrick® format. This system will be coupled to the ‘in‐house’ catabolic repression system (crp) generating energy efficient cell growth under glucose conditions as well as produce enzymes for hydrocarbon degradation when needed.

In summary, this approach will enable efficient hydrocarbon utilization under non-standard conditions. The chassis, in the form or BioBricks®, will facilitate the construction of strains with sustainable applications in the fossil fuel industry.