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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)
• E.coli K12 carrying [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] 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 characterization 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. The idea behind this is that E.coli might inherently contain an ADH and ALDH that, while it might be at an extremely low activity, can be able to degrade larger chain alkanes thus releasing energy for growth.

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. These assays will indicate the presence or absence of the desired enzymes, regardless of the alkane’s utilization for growth. 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 hydrocarbons from the medium using an apolar solvent (such as ethyl acetate) after the reaction and subsequent analysis by gas chromatography would indicate the presence of the corresponding alkanol and/or the decrease of alkane. For more on the experimental setup see the following pages:

• Resting-cell assays
• EtOAc extraction
• Gas chromatography program

[1] Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao. Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. Journal of molecular biology, 376: 453–465 (2008)

Characterization of the long-chain alkane monooxygenase LadA

Enzyme activity assay based on NADH absorbance

A less favored, yet very well accepted method for enzyme activity determination is by following the consumption of NADH at an absorption wavelength of 340 nm. By using a buffer at the appropriate pH, containing FMN and proper amounts of NADH the kinetics could easily be monitored by a 96-well plate reader. The protocol is described in detail here.

Enzyme activity assay based on GC-analysis

The most favorable way to analyse the activity of the LadA enzyme is by creating an environment in which it can properly function in-vitro. As explained earlier the enzyme required NADH and flavin mononucleotide as cofactors. Furthermore the optimal activity was found at a temperature of 60 degrees and a pH of 7.5. In accordance with these properties a protocol was set up for the conversion of hexadecane using the lysates of the E.coli cells carrying the ladA constructs. After the reaction the hydrocarbons are extracted with an apolar solvent and analysed by gas chromatography (click here for the GC program and the obtained GC calibration graph).

Characterization of the Alcohol DeHydrogenase (ADH) and ALdehyde DeHydrogenase (ALDH) systems

Growth on alcohols as sole carbon source

As in the case of alkane monooxygenases, our first attempt was to grow our recombinant strains on long-chain alcohols and aldehydes; hoping that E. coli will do the further degradation steps. For that purpose, we used octanol-1 and dodecanol-1 for the ADH system; whereas we used octanal and dodecanal for the ALDH system. If everything goes well, we should see microbial growth on minimal medium with these compounds as sole carbon source.

Resting cell assays

Our second attempt was to grow cells, until the exponential phase is reached; then the cells were collected and the spent medium was changed for a buffer with glucose that lacked of N-source, this was done in order to avoid more biomass production. Additionally we added alcohols and aldehydes to the mixture, so that if our part is working they will be converted in other molecules: alcohols to aldehydes and aldehydes to alkanoic acids.

With this approach, we are hoping that the enzymes produced during the growth phase will remain inside the cells. The glucose added will serve for maintenance purposes and co-factor regeneration. If everything works well, there will be some nice peaks appearing on our GC analysis.

NADH production in cell extracts

We called this assay: THE LAST RESORT =s

Basically, we grew cells until an O.D. (600nm) between 0.5-1.0. We needed chubby healthy happy exponential-growth cells because most of our constructions are meant for protein production in this growth phase.

Once, we got a lot of cells... we disrupted them by sonication and we analyze the NADH production by their guts using a simple spectrophotometrical analysis. If our parts are working, we should see a higher NADH production when the substrate (dodecanol-1 or dodecanal) to the buffer with microbial guts and NAD buffer. We can use this method because NAD is the natural co-factor of our proteins. Fortunately for us, NAD reduction could be easily quantified by absorbance measurements at 340 nm.

We called this protocol "the last resort" because the others didn't work. Which means that alcohols and aldehydes do not cross the cell membrane, thus in order to grow cell on these compounds as sole carbon sources... WE NEEDED TO ADD TRANSPORTERS =(