Summary

水性環境で炭化水素の変換を有効にするツールキット

Published: October 02, 2012
doi:

Summary

油汚染の浄化のための持続可能な自動調整システムは、標準的な細菌DNAの交換部品(BioBricks)を使用して設計されました。エンジニアリング<em> E大腸菌の</em>株が有毒、水性環境中のβ-酸化を介しアルカンを分解するために使用されていました。異なる種からそれぞれの酵素は、アルカン分解活性を示した。また、に耐性の増加<em> N</em>-ヘキサンをアルカントレラント細菌由来の遺伝子を導入することによって達成された。

Abstract

この作品は、前方の大腸菌によるアルカンの変換を可能にし、その適用性の原理の証明を提示するツールキットを置きます。このツールキットには、複数の標準交換部品(BioBricks)9アルカンの変換、有毒な炭化水素に富む環境における遺伝子発現と生存の調節をアドレッシングで構成されています。

アルカン分解の3段階経路を大腸菌で実装されました大腸菌それぞれアルカノール、カナール、最終的にアルカン-アミノ酸に、中長期鎖アルカンの変換を有効にする。後者はネイティブのβ-酸化経路を介して代謝された。中鎖アルカン(C5〜C13)とシクロアルカン(C5〜C8)、Gordonia spからアルカンヒドロキシラーゼシステムの4つの遺伝子(alkB2、rubA3、rubA4rubB)の酸化を促進する。 TF6 8,21を 大腸菌に形質転換した大腸菌 。の変換のための長鎖アルカン(C15-C36)は、Geobacillus thermodenitrificansからLADA遺伝子実装されました。分解プロセスの必要なさらなる手順については、ADHALDHは(G. thermodenitrificans由来する)10,11を導入ました。活動は休止細胞アッセイにより測定した。各酸化ステップでは、酵素活性が観察された。

プロセス効率を最適化するために、式は唯一、低グルコース条件下で誘導されました:基板-調節プロモーター、pCaiF、使用されていました。 pCaiFは大腸菌に存在している大腸菌 K12と非グルコース炭素源の分解に関与する遺伝子の発現を調節する。

ツールキットの最後の部分-生存を標的に- PhPFDαとβ溶媒耐性遺伝子、 パイロコッカスホリコシ OT3からの両方を使用して実装されていました。有機溶媒としては、否定的にaffectinによって細胞ストレスを誘導し、生存性を低下させる可能性がGタンパク質の折り畳み。シャペロンとして、PhPFDαβはアルカンの存在下でタンパク質の折りたたみのプロセスなどを改善します 。培地で10%n-ヘキサンのプレゼンスの増加成長率(最大50%)で示される炭化水素の改善·トレランスにつながったこれらの遺伝子の発現が観察された。

要約すると、結果は、ツールキットは大腸菌を可能にすること示す大腸菌水性環境中の炭化水素に変換して耐える。このように、それは合成生物学のアプローチを用いてオイル浄化のための持続的な解決に向けた第一歩を表しています。

Introduction

Oil pollution is among the most serious causes of environmental contamination, and greatly affects ecosystems, businesses and communities 3. Solutions are for example required to battle the continuous oil pollution originating from the oil sands tailing waters in Alberta, Canada. During the process of oil extraction from oil sands, bitumen, a semi-solid oxidized form of oil, is removed using thermal recovery techniques that consume about 3.1 barrels of water per single barrel of oil 1. Oil contaminated process water, mainly originating from a local river, is stored in tailing ponds after bitumen extraction. A more effective recycling of process water in order to reduce the need for freshwater uptake is needed. To facilitate the bitumen extraction and to ensure that downstream sites meet water quality guidelines for the protection of aquatic ecosystems, process water treatments are rapidly evolving 3.

To treat pollution of organic compounds, bioremediation technologies employing microorganisms are presently encouraged 1. Alkanes are the most abundant family of hydrocarbons in crude oil, containing 5 to 40 carbon atoms per molecule 7, 21. Many bacteria are known to degrade alkanes of various lengths via sequential oxidation of the terminal methyl group forming first alcohols, then aldehydes and finally fatty acids 8. Within this iGEM project several enzymes from different organisms were expressed and characterized, and made available via the BioBrick standard and Registry of Standard Biological Parts.

The well-studied alkane hydroxylase system of Gordonia sp. TF6 facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes using a minimum of four components: alkB2 (alkane 1-monooxygenase), rubA3, rubA4 (two rubredoxins) and RubB (rubredoxin reductase) 8, 21. Oxidation of long-chain alkanes (ranging from C15 up to C36) is reported to be performed by ladA, a flavoprotein alkane monooxygenase from Geobacillus thermodinitrificans NG-80-2 7, 15, 18, 22. LadA forms a catalytic complex with flavin mononucleotide (FMN) that utilizes atomic oxygen for oxidation. This results in the conversion of alkanes into the corresponding primary alkanol. The alcohols are further oxidized by alcohol and aldehyde dehydrogenases to fatty acids, which readily enter the β-oxidation pathway 7, 21. A zinc-independent alcohol dehydrogenase from the thermophillic bacterium Geobacillus thermoleovorans B23 oxidizes medium-chain alkanols into their respective alkanals, using NAD+ as a cofactor 10. Aldehyde dehydrogenase from the same bacterium is able to catalyze the NAD+-dependent final step in the medium-chain oxidation 11.

In order to reduce induction costs and to maintain optimal proliferation of the bacterial system, the promoter pCaiF from E.coli was characterized. This promoter can regulate expression of the hydrocarbon degradation pathway components, and is regulated by cAMP-Crp levels, which in turn depend on glucose levels 6. At high extracellular glucose concentrations in the environment the cellular cAMP (cyclic Adenosine Mononucleotide Phosphate) level was low through the inhibition of adenylyl cyclase as a side effect of PTS mediated glucose transport. Conversely, during limitation (low glucose concentrations) the cAMP level increased and Crp bound to cAMP forming the complex, cAMP-Crp, which bound pCaiF and activated transcription of the downstream components 6, 14.

Wildtype E. coli can only tolerate moderate concentrations of hydrocarbons. To complete the toolkit, tolerance to hydrocarbons had to be addressed. Several organic solvent-tolerant bacteria are known to survive in water-solvent two-phase systems 12. Molecular components known to increase tolerance are chaperones that facilitate the correct folding of proteins. The prefoldin system from Pyrococcus horikoshii OT3, consisting of the proteins phPFDα and phPFDβ, was shown to increase hydrocarbon-tolerance 17.

The alkane conversion toolkit was constructed following the BioBrick principle, which is documented at the Registry of Standard Biological Parts 9. BioBricks are plasmids containing a specific functional insert that is flanked by 4 predefined restriction sites. The BioBrick inserts can be extended flexibly, allowing the construction of biological systems with new functions.

Protocol

1。 BioBrickアセンブリ標準生物学的部分のレジストリからBioBricksはIGEM本部によって提供されています。既存BioBricksから新しいBioBrickを構築するために、アクセプター部分のドナー部分の下流に位置決めするための酵素EcoRIとSpeIでドナーBioBrick(最大1.0μg)を消化。アクセプター部の上流ドナー部分を位置決めするためのXbaIとPstIでダイジェスト。ドナーのバックボーンにカット第三適切…

Representative Results

Alkane conversion The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase. For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid BBa_…

Discussion

BioBrick原理は、アルカンの分解のために、シャーシを構築するために使われ、このツールキットの単一コンポーネントの原理の証明を得た。いくつかのアッセイは、in vivoおよびアルカン分解する経路酵素のin vitro活性を測定するために提案されている。提示された仕事は、正常宿主生物大腸菌の酵素活性と発現を決定するために使用できるメソッドの数を示しています適?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

このビデオの記事で行った実験国際遺伝子改変マシン競争9のために開発されました。著者はIGEMのチームメンバーにルークBergwerff、ピーテルTMバンBoheemen、Jelmerクノッセン、ヒューゴ·F Cuetoロハスとするためラモンファンデルファルクに感謝したいと思います研究の援助。我々は有用な議論ハン·デ·Winde、ステファンデコックとEsengülYILDIRIMに感謝し、この研究をホスティング。この作品は、バイオテクノロジーのデルフト工科大学部、デルフトインフォマティクス研究室、Bionanoscienceのデルフト工科大学学科、オイルサンドリーダーシップイニシアティブ(OSLI)、スタッドstudentenuitzendbureau、オランダゲノミクスイニシアティブ、Kluyverセンター、Nederlandse Vereniging Biotechnologische(開発元:Stichtingバイオオランダ)によってサポートされていました、DSM、GENEART、グライナーバイオ1とジェネンコア。

Materials

Name of the reagent Company Catalogue number Comments (optional)
E. coli K12 New England Biolabs C2523H  
Octane Fluka 74822  
Hexadecane Fluka 52209  
octanol-1 Fluka 95446  
dodecanol-1 Sigma-Aldrich 126799  
Hexane Sigma-Aldrich 296090  
NADH Sigma N4505  
FMN Sigma F2253  
MgSO4 J.T. Baker Casno 7487 889  
Triton X-100 Sigma-Aldrich T8787  
T4 ligase New England Biolabs M0202L  
Gas chromatograph      
Cell disrupter LA Biosystems CD-019  
Spectrophotometer Amersham pharmacia spec 2000  
Plate reader Tecan Magellan v7.0  
Incubator Innova, 44    
BioBrick K398014:
BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4– BBa_J61100-rubB
Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398014 Alkane Hydroxylase System
Resistance: Chloramphenicol
BioBrick K398027: BBa_R0040-BBa_B0034-ladA Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398027 ladA Protein Generator
Resistance: Chloramphenicol
BioBrick K398018: BBa_J23100-BBa_J61101-ADH Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398018 ADH generator
Resistance: Chloramphenicol
BioBrick K398030: BBa_R0040-BBa_B0034-ALDH Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398030 ALDH generator
Resistance: Chloramphenicol
BioBrick K398326: pCaiF Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398326 pCaiF promoter
Resistance: Chloramphenicol
BioBrick K398331: pCaiF-BBa_B0032-BBa_I13401 Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398331 pCaiF measurement device
Resistance: Chloramphenicol
BioBrick K398406: BBa_J23002-BBa_J61107-phPFDα-BBa_J61107- Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts BBa_K398406 Solvent tolerance cluster
Resistance: Chloramphenicol

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Cite This Article
Brinkman, E. K., Schipper, K., Bongaerts, N., Voges, M. J., Abate, A., Wahl, S. A. A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments. J. Vis. Exp. (68), e4182, doi:10.3791/4182 (2012).

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