En Toolkit til Aktiver carbonhydridomdannelse i vandige miljøer
Summary
En bæredygtig auto regulering bakterielt system til rensning af olie forureninger blev konstrueret ved hjælp af standard-udskiftelige DNA dele (BioBricks). Et konstrueret<em> E. coli</em> Stamme blev anvendt til at nedbryde alkaner via β-oxidation i toksiske vandige miljøer. De respektive enzymer fra forskellige arter viste alkan nedbrydningsaktivitet. Derudover en øget tolerance over<em> N</em>-Hexan blev opnået ved at indføre gener fra alkan-modstandsdygtige bakterier.
Abstract
Dette arbejde foreslås en værktøjskasse, der muliggør omdannelse af alkaner af Escherichia coli og præsenterer en proof of principle for dets anvendelighed. Værktøjskassen består af flere standard udskiftelige dele (BioBricks) 9 behandler konvertering af alkaner, regulering af genekspression og overlevelse i toksiske kulbrinte-rige miljøer.
En tre-trins vej for alkan nedbrydning blev gennemført i E. coli for at muliggøre omdannelse af mellem-og langkædede alkaner til deres respektive alkanoler, alkanals og i sidste ende alkan-syrer. Sidstnævnte blev metaboliseret via det native β-oxidation pathway. For at lette oxidation af mellemlange kæder alkaner (C5-C13) og cycloalkaner (C5-C8), fire gener (alkB2, rubA3, rubA4 og Rabb) af alkan-hydroxylase-systemet fra Gordonia sp. TF6 8,21 blev transformeret ind i E. coli. Til omdannelsen aflangkædede alkaner (C15-C36), blev LADA genet fra Geobacillus thermodenitrificans gennemført. For de nødvendige yderligere skridt i nedbrydningen processen blev ADH og ALDH (stammer fra G. thermodenitrificans) indført 10,11. Aktiviteten blev målt ved hvile celleassays. For hvert Oxidationen blev enzymaktivitet observeres.
For at optimere processen effektivitet blev ekspressionen kun induceres under lave glucosebetingelser: et substrat-reguleret promoter, pCaiF, blev anvendt. pCaiF er til stede i E. coli K12 og regulerer ekspressionen af generne involveret i nedbrydningen af ikke-glucose carbonkilder.
Den sidste del af toolkit – targeting overlevelse – blev gennemført ved hjælp af opløsningsmidler tolerance gener, PhPFDα og β, både fra Pyrococcus horikoshii OT3. Organiske opløsningsmidler kan fremkalde celle stress og reduceret overlevelsesevne ved negativt affecting proteinfoldning. Som chaperoner, forbedre PhPFDα og β proteinet foldeproces fx i nærværelse af alkaner. Ekspressionen af disse gener har ført til en forbedret carbonhydrid tolerance vist ved en forøget væksthastighed (op til 50%) i nærværelse af 10% n-hexan i dyrkningsmediet blev observeret.
Opsummerer de resultater indikerer, at toolkit muliggør E. coli til at konvertere og tolerere kulbrinter i vandige miljøer. Som sådan udgør det et første skridt hen imod en bæredygtig løsning for olie-oprydning ved hjælp af en syntetisk biologi tilgang.
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
The BioBrick princip anvendes til at konstruere et chassis for nedbrydningen af alkaner og en proof of principle for de enkelte komponenter i materialet blev opnået. Adskillige assays er foreslået at måle in vivo og di vitro-aktiviteten af alkan nedbrydende vej-enzymer. Den præsenterede arbejde med succes viser en række metoder, som kan anvendes til at bestemme enzymaktiviteter og ekspression i værtsorganismen E. coli efter implementering af egnede BioBricks. Det er endvidere vist,…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Eksperimenterne er udført i denne video-artikel blev udviklet til international gensplejsede Machine konkurrence 9. Forfatterne vil gerne takke IGEM teammedlemmer Luke Bergwerff, Pieter TM van Boheemen, Jelmer Cnossen, Hugo F. Cueto Rojas og Ramon van der Valk for bistanden i forskningen. Vi takker Han de Winde, Stefan de Kok og Esengül Yildirim for personer diskussioner og hosting denne forskning. Dette arbejde blev støttet af TU Delft Universitet Institut for Bioteknologi, The Delft Bioinformatics lab, TU Delft Institut for Bionanoscience, Oil Sands Leadership Initiative (OSLI), stud studentenuitzendbureau, Holland Genomics Initiative, Kluyver Centre, Nederlandse Biotechnologische Vereniging (Stichting Biotechnology Nederland) , DSM, Geneart, Greiner bio-one og 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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