En bærekraftig auto regulere bakteriell system for utbedring av oljeforurensninger designet ved hjelp av standard utskiftbare DNA deler (BioBricks). En konstruert<em> E. coli</em> Stamme ble brukt å degradere alkaner via β-oksidasjon i toksiske vandige miljøer. De respektive enzymer fra forskjellige arter viste alkan nedbrytningsaktiviteten. I tillegg kan en økt toleranse<em> N</em>-Heksan ble oppnådd ved å innføre gener fra alkan-tolerante bakterier.
Dette arbeidet legger frem en verktøykasse som gjør det mulig konvertering av alkaner av Escherichia coli og presenterer et bevis på prinsippet om anvendelse sin. Verktøysettet består av flere standard utskiftbare deler (BioBricks) 9 adressering konvertering av alkaner, regulering av genuttrykk og overlevelse i giftige hydrokarbon-rike miljøer.
En tre-trinns vei for alkan degradering ble implementert i E. coli å aktivere konvertering av middels og lang kjede alkaner til sine respektive alkanoler, alkanals og til slutt alkan-syrer. Sistnevnte ble metaboliseres via den innebygde β-oksidasjon veien. Å lette oksidasjonen av middels kjede alkaner (C5-C13) og sykloalkaner (C5-C8), fire gener (alkB2, rubA3, rubA4 og Rubb) av alkanet hydroksylase systemet fra Gordonia sp. TF6 8,21 ble forvandlet til E. coli. For konvertering avlangkjedede alkaner (C15-C36) ble LADA genet fra Geobacillus thermodenitrificans implementert. For de nødvendige ytterligere trinn for nedbrytning prosessen ble ADH og ALDH (som stammer fra G. thermodenitrificans) innført 10,11. Aktiviteten ble målt ved hvilende celle-analyser. For hvert oksidativ trinn ble enzymaktivitet observert.
Å optimalisere prosessen effektivitet, ble uttrykket bare indusert under lave glukose forhold: et substrat-regulert promotor, pCaiF, ble benyttet. pCaiF er tilstede i E. coli K12 og regulerer ekspresjon av gener som er involvert i nedbrytning av ikke-glukose karbonkilder.
Den siste delen av verktøysettet – targeting overlevelse – ble gjennomført ved hjelp av løsemidler toleranse gener, PhPFDα og β, både fra Pyrococcus horikoshii OT3. Organiske løsemidler kan indusere stress i cellene og redusert overlevelsesevne ved negativt affecting protein folding. Som anstand, PhPFDα og β forbedre proteinfolding prosessen f. eks under tilstedeværelsen av alkaner. Uttrykket av disse genene førte til en forbedret hydrokarbon toleranse vist ved en økt veksthastighet (opptil 50%) i nærværet av 10% n-heksan i dyrkningsmediet ble observert.
Oppsummering, tyder resultatene på at verktøysettet gjør E. coli å konvertere og tolerere hydrokarboner i vandige miljøer. Som sådan representerer den et første skritt mot en bærekraftig løsning for olje-utbedring ved hjelp av en syntetisk biologi tilnærming.
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.
Den BioBrick prinsippet brukes til å konstruere et chassis for nedbrytning av alkaner og et bevis prinsipp for enkeltstoffene av verktøysettet ble oppnådd. Flere analyser er foreslått å måle in vivo og in vitro aktivitet av alkan nedverdigende pathway enzymer. Den presenterte Arbeidet viser vellykket en rekke metoder som kan brukes til å bestemme på enzymaktiviteter og uttrykk i vertsorganismen E. coli etter innføringen av egnede BioBricks. Videre er det vist at BioBrick prinsippet ka…
The authors have nothing to disclose.
Eksperimenter utført i denne video-artikkelen ble utviklet for det internasjonale genmodifisert Machine konkurranse 9. Forfatterne ønsker å takke iGEM gruppemedlemmer Luke Bergwerff, Pieter TM van Boheemen, Jelmer Cnossen, Hugo F. Cueto Rojas og Ramon van der Valk for hjelp i forskningen. Vi takker Han de Winde, Stefan de Kok og Esengül Yıldırım for personer diskusjoner og hosting denne forskningen. Dette arbeidet ble støttet av TU Delft University Institutt for bioteknologi, The Delft Bioinformatikk lab, TU Delft Institutt for Bionanoscience, Oil Sands Leadership Initiative (OSLI), stud studentenuitzendbureau, Nederland Genomics Initiative, Kluyver Centre, Nederlandse Biotechnologische Vereniging (Stichting Bioteknologi Nederland) , DSM, Geneart, Greiner Bio-One og Genencor.
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 |