수성 환경에서 탄화수소 전환을 사용하는 툴킷
Summary
오일 오염의 개선을 위해 박테리아 시스템을 규제 지속 가능한 자동차는 표준 호환 DNA 부품 (BioBricks)를 사용하여 설계되었습니다. 엔지니어링<em> E. 대장균</em> 변형은 독성 수성 환경에서 β-산화를 통해 alkanes을 저하하는 데 사용되었다. 종에서 각각의 효소는 alkane 저하 활동을 보여 주었다. 에 또한 증가 허용<em> N</em> – 헥산은 alkane에 강한 박테리아 유전자를 도입하여 달성되었다.
Abstract
이 작품은 앞으로 대장균에 의해 alkanes의 전환을 가능하게하고 적용의 원리의 증명을 제시하는 툴킷를 게재 할 수 있습니다. 툴킷은 여러 표준 교환 부품 (BioBricks) 9 alkanes의 전환, 독성 탄화수소 풍부한 환경에서의 유전자 발현과 생존의 규정을 해결로 구성되어 있습니다.
alkane 저하에 대해 3 단계 경로는 E.에 구현 된 각 alkanols, alkanals 궁극적으로 alkanoic – 산에 중간과 긴 체인 alkanes의 전환을 활성화 대장균. 후자는 기본 β-산화 경로를 통해 대사되었다. 중간 체인 alkanes (C5-C13)와 cycloalkanes (C5-C8), Gordonia SP에서 alkane hydroxylase 시스템 네 가지 유전자 (alkB2, rubA3, rubA4 및 rubB)의 산화를 촉진합니다. TF6 8,21는 E.로 변환 된 대장균. 의 전환에 대한장쇄 alkanes (C15-C36)는 Geobacillus thermodenitrificans에서 라다 유전자가 구현되었습니다. 열화 과정에 필요한 자세한 단계를 들어, ADH 및 ALDH은 (G. thermodenitrificans에서 발생) 10,11을 도입했다. 활동은 휴식 세포 assays에 의해 측정되었다. 각 산화 단계에 효소 활동이 관찰되었다.
프로세스 효율을 최적화하려면, 그 표현은 낮은 포도당 조건에서 유도되었습니다 기판 – 규제 발기인, pCaiF가 사용되었습니다. pCaiF는 E.에 존재 대장균 K12 및 비 포도당 탄소 소스의 저하에 관련된 유전자의 표현을 규제하고있다.
툴킷의 마지막 부분 – 생존을 대상으로는 – PhPFDα와 β 용매 내성 유전자, Pyrococcus horikoshii OT3에서 모두를 사용하여 구현되었습니다. 유기 용제에 부정적인 affectin에 의해 세포 스트레스를 유발하고 survivability을 감소 할 수 있습니다g 단백질 폴딩. 보호자로서 PhPFDα와 β는 alkanes의 존재에 따라 단백질 접힘 과정 등을 향상시킵니다. 문화 매체에서 10 % N-헥산의 presences의 증가 성장 속도 (최대 50 %)로 표시 개선 탄화수소 허용으로 이어이 유전자의 표현이 관찰되었다.
요약, 결과는 툴킷은 E. 수있게 된 것을 나타냅니다 수성 환경에서 탄화수소를 변환하고 관대 할 수 대장균. 따라서, 그것은 합성 생물학 접근 방식을 사용하여 오일 개선을위한 지속 가능한 솔루션에 대한 초기 단계를 나타냅니다.
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
Representative Results
Discussion
BioBrick의 원리는 alkanes의 저하에 대한 섀시를 만드는 데 사용되며 툴킷의 단일 구성 요소에 대한 원칙의 증명 얻은 것입니다. 여러 assays는 생체과 alkane 굴욕 경로의 효소의 체외 활동에 측정하기 위해 제안합니다. 제시 작업은 성공적으로 호스트 유기체 E.의 효소 활동과 표현을 결정하는 데 사용할 수있는 방법의 수를 보여줍니다 적절한 BioBricks를 구현 한 후 대장균. 또…
Disclosures
The authors have nothing to disclose.
Acknowledgements
이 비디오 문서에 수행 실험은 국제 유전 공학 기계 대회 9 개발되었다. 저자는 대한 iGEM 팀 회원 눅 Bergwerff, 에테르 TM 반 Boheemen, Jelmer Cnossen, 휴고 F. 쿠에 토 로자 스와 라몬 반 데르 Valk을 감사드립니다 연구 지원. 우리는 도움이 토론이 연구를 호스팅 한 데 Winde, 스테판 드 콕과 Esengül Yıldırım 감사드립니다. 이 작품은 생명 공학의 TU 델프트 대학과, 델프트 생물 정보학 연구실, Bionanoscience의 TU 델프트 (Delft)과, 오일 샌드 리더십 이니셔티브 (OSLI), 스터드 studentenuitzendbureau, 네덜란드 유전체학 이니셔티브, Kluyver 센터, Nederlandse Biotechnologische Vereniging (에는 Stichting 생명 공학 네덜란드)에 의해 지원되었다 , DSM, Geneart, Greiner 바이오 하나 Genencor.
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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