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From 2010.igem.org

Alkane Degradation Characterization

Introduction

In summary, the following strains were characterized:

• E.coli K12 carrying [http://partsregistry.org/Part:BBa_K398014 BBa_K398014] in pSB1A2 (AH system)
• E.coli K12 carrying [http://partsregistry.org/Part:BBa_K398017 BBa_K398017] in pSB1A2 (LadA)
• E.coli TOP10 carrying [http://partsregistry.org/Part:BBa_K398027 BBa_K398027] in pSB1A2 (LadA)
• E.coli K12 carrying [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] in pSB1A2 (ADH)
• E.coli K12 carrying [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] in pSB1A2 (ALDH)

The characterization will generally be executed along with an 'empty' plasmid carrying strain:

• E.coli K12 carrying [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] in pSB1A2 ('empty' plasmid)

Characterization of the alkane hydroxylase system

Growth analysis

Of course, one of the first characterizzation experiments was to test growth of the E.coli strains carrying BBa_K398014 on alkanes. The alkanes octane and dodecane were tested as possible substrates in M9 minimal medium. The protocol can be found found [here]

Resting-cell assays

As explained earlier the catalytic component of the alkane hydroxylase system is an integral membrane protein. Characterization must thus be done using an intact-membrane setup. An option which has been explored in literature [1] is the resting-cell assay a.k.a. biotransformation assay. The logic behind this is to stall the growth of a large volume of cells by using nitrogen-deficient medium to test their alkane conversion capabilities at near-zero growth. Extraction of the medium using an apolar solvent (such as ethyl acetate) after a certain amount of time and subsequent analysis by gas chromatography would indicate the presence of the corresponding alkanol and the decrease of alkane. For more on the experimental setup click [here], for the extraction method [here] and for the gas chromatography program [here].

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.