Team:TU Delft/Project/alkane-degradation/characterization


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Characterization of the Alkane Degradation Parts

The following strains will be characterized:

  • E.coli K12 containing BBa_K398028 in pSB1A2 (AlkB and RubA3) and BBa_K398011 in pSB1C3 (RubR and RubA4)
  • E.coli K12 containing BBa_K398017 in pSB1A2 (LadA)
  • E.coli K12 containing BBa_K398026 in pSB1A2 (ADH and ALDH)
  • E.coli K12 containing BBa_K398026 in pSB1A2 (ADH and ALDH) and BBa_K398017 in pSB1C3 (LadA)
  • E.coli K12 containing an ‘empty’ pSB1A2 plasmid

The characterization will involve three main aspects: 1. The alkane lengths that are converted by the respective genes 2. The speed at which the alkanes are converted by the enzymes 3. Possible growth of E.coli K12 on the respective alkane

Characterization of growth

The preliminary characterization will aim to determine the presence of growth on any one of the following alkanes:

  • octane (C8)
  • undecane (C11)
  • dodecane (C12)
  • tetradecane (C14)
  • heptadecane (C17)
  • octadecane (C18)

For the growth characterization on short-chain and long-chain alkanes an E.coli K12 strain containing BBa_K398015 and BBa_K398022 (or intermediates thereof) is inoculated into M9 minimal medium containing 5% v/v ratio of the respective alkane on a 96-well plate with 200 μL volume per well. Growth will be determined o/n by absorbance at 600nm with intervals of 10 minutes.

Characterization of enzyme functionality

Parallel to this, resting-cell assays will be performed on growth-inhibited E.coli K12 strains containing the constructs described earlier. These assays will indicate the presence or absence of the desired enzymes, regardless of the alkane’s utilization for growth. The hydrocarbon compositions will be determined by gas chromatography analysis. For the intracellular enzymes, the cells were lysed and treated with the appropriate akanols and alkanals.