एक स्थायी तेल प्रदूषण के remediation के लिए जीवाणु प्रणाली को विनियमित ऑटो मानक विनिमेय डीएनए भागों (BioBricks) का उपयोग कर बनाया गया था. एक इंजीनियर<em> ई. कोलाई</em> तनाव विषाक्त वातावरण में जलीय β ऑक्सीकरण के माध्यम से alkanes नीचा करने के लिए इस्तेमाल किया गया था. विभिन्न प्रजातियों में से संबंधित एंजाइमों alkane गिरावट गतिविधि दिखाया. इसके अतिरिक्त, एक वृद्धि की सहिष्णुता<emN></em> Hexane alkane सहिष्णु बैक्टीरिया के जीन से शुरू करने से हासिल की थी.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit – targeting survival – was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
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.
BioBrick सिद्धांत alkanes की गिरावट के लिए एक हवाई जहाज़ के पहिये का निर्माण करने के लिए प्रयोग किया जाता है और टूलकिट के एक घटक के लिए सिद्धांत का एक सबूत प्राप्त किया गया था. कई assays के लिए vivo में और alkane अपमानजनक म?…
The authors have nothing to disclose.
इस वीडियो लेख में प्रदर्शन प्रयोगों अंतरराष्ट्रीय आनुवंशिक रूप से इंजीनियर मशीन 9 प्रतियोगिता के लिए विकसित किए गए लेखकों iGEM टीम के सदस्यों के लिए ल्यूक Bergwerff, पीटर टीएम वैन Boheemen, Jelmer Cnossen, ह्यूगो एफ Cueto रोजास और Ramon वैन der Valk का शुक्रिया अदा करना चाहते हैं अनुसंधान में सहायता. हम उपयोगी विचार – विमर्श और इस शोध की मेजबानी के लिए हान डी winde, Stefan De Kok और Esengül Yildirim धन्यवाद. यह काम टीयू डेल्फ़्ट विश्वविद्यालय जैव प्रौद्योगिकी विभाग, डेल्फ़्ट बायोइनफॉरमैटिक्स प्रयोगशाला, टीयू डेल्फ़्ट Bionanoscience के विभाग, तेल रेत नेतृत्व पहल (OSLI), स्टड studentenuitzendbureau, नीदरलैंड जीनोमिक्स पहल, Kluyver केंद्र, Nederlandse Biotechnologische Vereniging (Stichting बायोटैक्नोलॉजी Nederland) द्वारा समर्थित किया गया , DSM, Geneart, Greiner जैव एक और 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 |