En verktygslåda så att kolväteomvandling i vattenhaltiga miljöer
Summary
En hållbar automatisk reglering bakteriell system för sanering av olja föroreningar ritades med vanliga utbytbara DNA delar (BioBricks). Konstruerad<em> E. E. coli</em> Stam användes för att bryta ned alkaner via β-oxidation i toxiska vattenhaltiga miljöer. De respektive enzymer från olika arter visade alkan nedbrytning aktivitet. Dessutom, en ökad tolerans för<em> N</em>-Hexan uppnåddes genom att införa gener från alkan-toleranta bakterier.
Abstract
Detta arbete lägger fram en verktygslåda som möjliggör omvandlingen av alkaner av Escherichia coli och presenterar ett bevis på principen för dess tillämplighet. Verktygslådan består av flera vanliga utbytbara delar (BioBricks) 9 behandlar omvandlingen av alkaner, reglering av genuttryck och överlevnad i giftiga kolväterika miljöer.
Ett tre steg väg för alkan nedbrytning genomfördes i E. E. coli för att möjliggöra omvandlingen av medellång och lång kedja alkaner till deras respektive alkanoler, alkanals och i slutändan alkansyra-syror. De sistnämnda metaboliseras via nativa β-oxidation vägen. För att underlätta oxidationen av medellånga kedja alkaner (C5-C13) och cykloalkaner (C5-C8), fyra gener (alkB2, rubA3, rubA4 och Rubb) i alkan hydroxylas systemet från Gordonia sp. TF6 8,21 transformerades in i E. coli. För omvandling avlångkedjiga alkaner (C15-C36), fick lada genen från Geobacillus thermodenitrificans genomförts. För de nödvändiga ytterligare steg i nedbrytningsprocessen fick ADH och ALDH (ursprung från G. thermodenitrificans) infördes 10,11. Aktiviteten mättes genom vilande cellanalyser. För varje oxidativa steget, var enzymaktiviteten observerades.
För att optimera processens effektivitet, var uttrycket endast induceras under låga glukos betingelser: ett substrat-reglerad promotor, pCaiF, användes. pCaiF finns i E. coli K12 och reglerar uttrycket av gener som är involverade i nedbrytningen av icke-glukos kolkällor.
Den sista delen av verktygslådan – inriktning överlevnad – genomfördes med användning av lösningsmedel tolerans gener, PhPFDα och β, båda från Pyrococcus horikoshii OT3. Organiska lösningsmedel kan inducera cell-stress och minskad överlevnad med negativt affecting. proteinveckning. Som chaperones, PhPFDα och β förbättra proteinveckning processen t.ex. under närvaron av alkaner. Uttrycket av dessa gener ledde till en förbättrad kolväte tolerans visas genom en ökad tillväxthastighet (upp till 50%) i närvaro av 10% n-hexan i odlingsmediet observerades.
Sammanfatta, tyder resultaten på att det verktygslådan gör E. E. coli att konvertera och tolerera kolväten i vattenbaserade miljöer. Som sådan utgör den ett första steg mot en hållbar lösning för olje-sanering med hjälp av en syntetisk biologi strategi.
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
Den BioBrick Principen används för att konstruera ett chassi för nedbrytning av alkaner och ett bevis på principen för de enskilda komponenterna i verktygslådan erhölls. Flera analyser föreslås att mäta in vivo-och in vitro-aktivitet av alkan nedbrytande enzymer pathway. Den presenterade arbete visar framgångsrikt ett antal metoder som kan användas för att bestämma enzymaktiviteter och uttryck i värdorganismen E. E. coli efter genomförandet av lämpliga BioBricks. Vidare visas det att B…
Disclosures
The authors have nothing to disclose.
Acknowledgements
De experiment som utförts i denna video-artikeln har utvecklats för den internationella Genmanipulerade Maskin tävling 9. Vill tacka iGEM gruppmedlemmar Luke Bergwerff, Pieter TM van Boheemen, Jelmer Cnossen, Hugo F. Cueto Rojas och Ramon van der Valk för författarna stödet i forskningen. Vi tackar Han de Winde, Stefan de Kok och Esengül Yıldırım för hjälp diskussioner och hosting denna forskning. Detta arbete stöddes av TU Delft University Institutionen för bioteknik, Delft Bioinformatics lab, TU Delft Institutionen för Bionanoscience, Oil Sands Ledarskap Initiative (OSLI), Stud studentenuitzendbureau, Nederländerna Genomics Initiative, Kluyver Centre Nederlandse Biotechnologische Vereniging (Stichting Biotechnology Nederland) , DSM, Geneart, Greiner Bio-One och 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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