Team:TU Delft/Project

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Project Abstract

Alkanivore: Enabling hydrocarbon degradation in aqueous environments

Pollution of soil and water environments by crude oil has been, and is still today, an important environmental issue. This was once more confirmed with the oil-spill in the Gulf of Mexico, but is also an issue that has to be faced continuously during the process of oil extraction from oil sands. Cleaning has proven to be challenging, but synthetic biology may hold the key to sustainable bio-remedial solutions for the future. What if we could design a small, autonomous, self-replicating, inexpensive method to remove oil from aqueous environments? The TU Delft iGEM 2010 team spent their summer designing a system that can tolerate, sense, dissolve & degrade hydrocarbons in aqueous environments, which could open new doors for the oil-industry.

Read our full introduction

Sub projects

The basis of the 2010 TU Delft iGEM team's project is the generation of a biological chassis for the conversion of hydrocarbons. The conversion system will be implemented and characterized using the well-studied cellular environment of Escherichia coli, the workhorse for genetic and metabolic engineering. To tackle the important aspects faced when using biological systems for oil utilization, we are focusing on the following features:

Alkane degradation

The aerobic alkane conversion pathways for various lengths of alkanes from Gordonia sp. TF6 and Geobacillus thermodenitrificans were the basis for the alkane degradation parts. These pathways were implemented in E.coli using the BioBrick principle and characterized in detail with respect to single enzyme activities and affinity. Using these measurements the efficiency of different enzymes can easily be compared. We succesfully characterized the alkane hydroxylase system (octane conversion), which had a 30-fold increase in enzymatic activity compared to the negative control. Furthermore, the enzyme activity of E.coli lysates was found to be at least 6-fold higher on hexadecane when the long-chain alkane monooxygenase (ladA) protein was expressed. The characterization showed that the implemented alcohol dehydrogenase (ADH, for the conversion of long chain alkanols to alkanals) converted dodecanol 43% better than the standard E.coli. On the other hand, the expression of our ALDH system increased the dodecanal dehydrogenase activity in E.coli cell extracts by 2-fold, which was around 34% of the total aldehyde dehydrogenase activity for our positive control Pseudomonas putida a natural alkane-degrading bacterium.

The degradation pathways were also included in a Metabolic Flux Analysis together with possible valuable products. More information on this can be found on the in silico MFA page

Sensing

In order to have efficient cell growth, it is important to develop a system that activates gene expression at the optimal moment in time. Parts were designed based on an alkane sensing mechanism from Pseudomonas putida, in which a number of different promoters are activated by the presence of alkanes or the absence of glucose. Characterization of one of these promoters resulted in production of GFP at low glucose concentrations. This new promoter combined with B0032 has a GFP production rate of 3.975E07 GFP molecules/second/O.D. in the stationary phase (glucose starvation). We also found that the same part [http://partsregistry.org/Part:BBa_K398326 pCaiF] is nicely tuned by intracellular cAMP levels. Furthermore this system was also modeled, more information can be found on the in silico sensing page

Survival

In order to be able to use our alkane degrading biological system in the intended environments some though must go into the survivability of the system. These environments include those with high levels of hydrocarbons as well as high salt levels, aspects which would cause an inhabitable environment for our system. It was found that organisms which are naturally hydrocarbon tolerant and salt tolerant produce chaperons and other proteins, which maintain the cellular activity - these genes were the basis of the survival parts. Characterization of these parts showed an increased survivability for both salt and hydrocarbon environments of up to 33% and 50% respectively.

Read more about Salt & Alkane Tolerance.

Solubility

An engineering challenge faced in oil bio-conversions is the low solubility of hydrophobic molecules in water. To overcome this mass‐transfer limitation, a gene encoding for AlnA, a protein with emulsifying properties, was the basis of our solubility BioBrick. Currently described protocols deemed insufficient to determine the solubility, thus a new protocol was created to characterize this part, which showed an increased solubility of about 20%.

RBS Characterization

In order to be able to tune our protein production, and for the benefit of the iGEM community, the Anderson RBS family was characterized. The parts were build up out of a standard promoter, the to be characterized RBS and GFP as a a signal molecule. By measuring the GFP production in the exponential phase the individual strengths of the different RBS's were determined

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-The aerobic alkane conversion pathways for various lengths of alkanes from ''Gordonia sp. TF6'' and ''Geobacillus thermodenitrificans'' were the basis for the [[Team:TU_Delft/Project/alkane-degradation/parts|alkane degradation parts]]. These pathways were implemented in ''E.coli'' using the BioBrick principle and [[Team:TU_Delft/Project/alkane-degradation/characterization|characterized]] in detail with respect to single enzyme activities and affinity. Using these measurements the efficiency of different enzymes can easily be compared. '''The characterization showed that'''
+The aerobic alkane conversion pathways for various lengths of alkanes from ''Gordonia sp. TF6'' and ''Geobacillus thermodenitrificans'' were the basis for the [[Team:TU_Delft/Project/alkane-degradation/parts|alkane degradation parts]]. These pathways were implemented in ''E.coli'' using the BioBrick principle and [[Team:TU_Delft/Project/alkane-degradation/characterization|characterized]] in detail with respect to single enzyme activities and affinity. Using these measurements the efficiency of different enzymes can easily be compared.
-the implemented alcohol dehydrogenase (ADH, for the conversion of long chain alkanols to alkanals) converted dodecanol 43% better than the standard ''E.coli''. On the other hand, the expression of our ALDH system increased the Dodecanal dehydrogenase activity in ''E.coli'' cell extracts by 2-fold, which was around 34% of the total aldehyde dehydrogenase activity for our positive control ''Pseudomonas putida'' a natural alkane-degrading bacterium.
+We succesfully characterized the alkane hydroxylase system (octane conversion), which had a 30-fold increase in enzymatic activity compared to the negative control. Furthermore, the enzyme activity of ''E.coli'' lysates was found to be at least 6-fold higher on hexadecane when the long-chain alkane monooxygenase (ladA) protein was expressed. The characterization showed that the implemented alcohol dehydrogenase (ADH, for the conversion of long chain alkanols to alkanals) converted dodecanol 43% better than the standard ''E.coli''. On the other hand, the expression of our ALDH system increased the dodecanal dehydrogenase activity in ''E.coli'' cell extracts by 2-fold, which was around 34% of the total aldehyde dehydrogenase activity for our positive control ''Pseudomonas putida'' a natural alkane-degrading bacterium.
 The degradation pathways were also included in a [[Team:TU_Delft/Modeling/MFA/explanation|Metabolic Flux Analysis]] together with possible valuable products. More information on this can be found on the [[Team:TU_Delft/Modeling/MFA|in silico MFA page]]