Difference between revisions of "Part:BBa K398014"

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Alkane Hydroxylase System

Figure 1:Complete Alkane degradation pathway, AlkB is one of the 1st steps herein

Introduction

The alkane hydroxylase system from Gordonia sp. TF6 facilitates the initial step of the degradation of C⁵-C¹³ alkanes as well as that of C⁵-^C8 cycloalkanes towards their respective alcohols. Based on the literature on this topic it is expected that the in-house mechanism of E.coli will be able to further degrade the products of this pathway.

The AH construct consists of the sequences encoding for:

• Alkane 1-monooxygenase (alkB2); an integral cytoplasmic membrane monooxygenase of which homologs have been reported for varying genus and species. This is the catalytic component of the AH system and as such oxidizes (cyclo)alkanes to their respective (cyclo)alkanols by transferring one oxygen atom from molecular oxygen to the alkane.
• Rubredoxin reductase (rubB); catalyzes the reduction of the second oxygen atom released from molecular oxygen using electrons supplied by NADH.
• Rubredoxin (rubA3); facilitates the transfer of electrons from NADH to rubredoxin reductase.
• Rubredoxin (rubA4); an electron-carrier protein required by the AH system.

The AH construct was designed to harbor all four of the required coding sequences -each behind its own RBS region- sharing a constitutive promoter.

Characterization

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 [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Protocol_for_growth_of_E.coli_K12_on_alkanes 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:

• [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli Resting-cell assays]
• [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol EtOAc extraction]
• [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols Gas chromatography program]

Results

References

1. Fujii, T., Narikawa, T., Takeda, K., Kato, J., Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6. Bioscience, biotechnology, and biochemistry, 68(10) 2171-2177 (2004)
2. 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)

Sequence and Features