一个工具包，使水环境中的烃类转化
Summary
一个可持续发展的自动调节修复石油污染的细菌系统的设计采用标准的可互换的DNA部分（BioBricks）。一个工程<em> E.大肠杆菌</em>菌株用于在有毒的水溶液环境中，通过β-氧化降解烷烃。来自不同物种的酶显示烷烃的降解活性。此外，增加的耐受性<em> N</em>己烷，通过引入从烷烃耐受菌的基因来实现。
Abstract
这项工作提出了一个工具包，使烷烃转化大肠杆菌 ，并提出证明其适用的原则。该工具包包括多个标准的可互换的部分（BioBricks）的9解决烷烃转化，调节基因的表达和生存有毒碳氢化合物丰富的环境。
E.实施三个步骤的烷烃降解途径大肠杆菌介质和长链烷烃的转化率，以使各自的链烷醇，烷醛和最终链烷酸。后者则是通过本机的β-氧化途径代谢。为了方便的中链烷烃的（C5-C13）和环烷烃（C5-C8），4个基因（alkB2，rubA3 rubA4和擦除 ）的烷烃羟化酶系统从大头属的氧化。 TF6 8,21转化到大肠杆菌大肠杆菌 。为转换的长链烷烃（C15-C36）， 芽孢杆菌热拉达基因的实施。对于需要采取进一步措施的退化过程，ADH和ALDH（G.热 ）的10,11。活性测定采用静息细胞检测。对于每个氧化工序中，酶的活性进行了观察。
为了优化流程的效率，表达诱导低血糖条件下基板调节的启动，pCaiF，。 pCaiF是在E.大肠杆菌 K12和调节非葡萄糖碳源的降解中所涉及的基因的表达。
使用的工具包-针对生存的最后一部分-溶剂耐受的基因，PhPFDα和β，无论是从激烈热horikoshii OT3。有机溶剂可以诱导细胞应激和下降的生存能力产生负面affectin克蛋白质折叠。作为分子伴侣，PhPFDα和β改善蛋白质的折叠过程中， 例如，链烷烃的存在下。所示的一种改进的烃类公差在培养基中的增加的生长率（高达70％）10％n-己烷的存在导致这些基因的表达进行了观察。
总之，结果表明，该工具包能够E.大肠杆菌转换和容忍在水环境中的碳氢化合物。因此，它代表了一个可持续发展的解决方案，油整治利用合成生物学的方法的第一步。
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
BioBrick原则被用于构造的底架的与链烷烃的降解和该工具包为单个元件的一个原则的证明得到。几个实验提出在体内和体外活性烷烃降解途径酶的测量。所提出的工作成功地演示了一些方法，这些方法可以被用来确定酶的活性和表达在宿主有机体E.大肠杆菌合适的BioBricks后实施。此外，它是示出，该BioBrick原则可以用来设计一个有机体表达蛋白质所需的链烷烃的降解，提供了一个…
Disclosures
The authors have nothing to disclose.
Acknowledgements
在这个视频文章进行的实验，开发了国际基因机器设计竞赛9。想感谢iGEM比赛的团队成员，卢克Bergwerff，彼得TM面包车Boheemen，Jelmer克诺森，乌戈·F.奎托·罗哈斯和拉蒙·Van Der Valk酒店协助这项研究。我们感谢韩Winde，斯特凡去角和Esengül的的耶尔德勒姆的有益讨论和主持这项研究。这项工作是支持的代尔夫特理工大学生物技术系，生物信息学实验室的代尔夫特，代尔夫特理工大学的Bionanoscience，油砂领导倡议（OSLI），梭哈studentenuitzendbureau，荷兰基因组计划，Kluyver中心，荷兰Biotechnologische Vereniging（STICHTING生物技术荷兰） ，DSM，香港传艺，格雷纳生物和杰能科。
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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