The experiments performed in this video-article were developed for the international Genetically Engineered Machine competition 9.The authors would like to thank iGEM team members Luke Bergwerff, Pieter T.M. van Boheemen, Jelmer Cnossen, Hugo F. Cueto Rojas and Ramon van der Valk for the assistance in the research. We thank Han de Winde, Stefan de Kok and Eseng&uuml;l Y&#305;ld&#305;r&#305;m for helpful discussions and hosting this research. This work was supported by the TU Delft University Department of Biotechnology, The Delft Bioinformatics lab, TU Delft Department of Bionanoscience, Oil Sands Leadership Initiative (OSLI), StuD studentenuitzendbureau, Netherlands Genomics Initiative, Kluyver Centre, Nederlandse Biotechnologische Vereniging (Stichting Biotechnology Nederland), DSM, Geneart, Greiner bio-one and Genencor.

1. BioBrick Assembly
<ol>
 <li>BioBricks from the Registry of Standard Biological parts are provided by iGEM headquarters. To construct a new BioBrick from existing BioBricks, digest the donor BioBrick (up to 1.0 &mu;g) with the enzymes EcoRI and SpeI for positioning the donor part downstream of the acceptor part. Digest with XbaI and PstI for positioning the donor part upstream of the acceptor part. Add a third appropriate restriction enzyme that cuts in the backbone of the donor. Perform the digestions in a total volume of 20-25 ml with the appropriate buffer, according to the supplier (final concentration 1x). Use 5 units/&mu;g DNA for the restriction enzymes.</li>
 <li>Digest the acceptor BioBrick with either EcoRI and XbaI or SpeI and PstI.</li>
 <li>Incubate the digestions for (at least) one hr at 37 &deg;C. Inactivate the restriction endonucleases by heat, incubation at 80 &deg;C for 10 min and centrifuge shortly.</li>
 <li>Ligate the digested BioBrick parts (donor and acceptor) together. Since XbaI and SpeI generate compatible DNA ends, a mixed site is created that cannot be cut with any restriction enzyme resulting in a new 'combined' BioBrick that flanked by the 4 standard restriction sites. In the ligation mixture the final DNA concentration is preferably ~100 ng/&mu;l. Perform the ligation reaction in a total volume of 10-15 ml with the T4 ligation buffer (final concentration 1x) and T4 ligase (1 unit/&mu;g DNA).</li>
 <li>Incubate the ligation mixture at 16 &deg;C for at least 3 hr.</li>
 <li>Perform transformation reaction with circa half of the ligation mix.</li>
 <li>Confirm the BioBrick to be correct with sequencing.</li>
 <li>To construct a new BioBrick from synthesized DNA, modify the genes for synthesis for optimal expression in E. coli by means of the JCat website tool (<a href="http://www.jcat.de/" target="_blank">http://www.jcat.de/</a>). Make further modifications in accordance with the requirements of the BioBrick standard. This standardization implies that every BioBrick is composed of a DNA sequence of interest preceded by a prefix and followed by a suffix. The prefix and suffix are sequences that contain predefined restriction sites, that are absent in the remaining plasmid sequence. These standard restriction sites make it possible to interchange and extend the BioBrick inserts flexibly 9. The prefix and suffix have the following sequences:</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Sequence</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Prefix</td>
 <td>5' GAATTCGCGGCCGCTTCTAG3'</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>5' GAATTCGCGGCCGCTTCTAGAG 3'</td>
 <td>If the following part is a coding sequence or any part that starts with "ATG"</td>
 </tr>
 <tr>
 <td>Suffix</td>
 <td>5' TACTAGTAGCGGCCGCTGCAG 3'</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
2. Alkane Conversion Resting Cell Assay, In Vivo
This assay was performed based on the method described by Fujii et al. (2004).
<ol>
 <li>Culture E. coli cells expressing the Alkane Hydroxylase system (<a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a>) and cells carrying an empty vector (<a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a>) in 5 ml LB medium with appropriate antibiotics overnight.</li>
 <li>Transfer 500 &mu;l of the overnight culture into 50 ml of fresh LB (with antibiotic) and incubate until the cell turbidity reached an OD (optical density) of 0.3 at 600 nm.</li>
 <li>Centrifuge for 10 min at 4,000 rpm and resuspend the pellet in 5 ml 0.1 M phosphate buffer (pH 7.4).</li>
 <li>Centrifuge again for 10 min at 4,000 rpm and resuspend the pellet in 5 ml 0.1 M phosphate buffer now containing E2 salts and 0.66% v/v glycerol (nitrogen-deficient medium).</li>
 <li>Measure the cell turbidity (OD600).</li>
 <li>Prepare cell-mixture aliquots of 6 ml in 25 ml closed-cap glass flasks and no-cell controls (E2 salts + 0.66% v/v glycerol).</li>
 <li>Add 100 nmol of alkane to each flask.</li>
 <li>Incubate the mixtures at 37 &deg;C for 24 hr (shorten when higher rates are obtained).</li>
 <li>Measure the OD600 after incubation.</li>
 <li>Extract the hydrocarbons in the culture media by ethyl acetate and determine hydrocarbon concentration in cell culture by gas chromatography (see protocol 3).</li>
 <li>Calculate the degradation per unit of biomass by dividing the total amount of alkane converted by the total biomass present in each flask (convert OD600 to dry weight). Dividing the result by the experimental duration yields the degradation activity per unit biomass per time unit. The average is taken from three individual runs.</li>
</ol>
3. Alkane Conversion Enzyme Assay, In Vitro
This assay was performed essentially according to the method described by Li et al. (2008).
<ol>
 <li>Culture E. coli cells expressing the ladA gene (<a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a>) and cells carrying an empty vector (<a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a>) in 50 ml LB medium with appropriate antibiotics overnight.</li>
 <li>Transfer 500 &mu;l of the overnight culture into 50 ml of fresh LB (with antibiotic) and incubate until the cell turbidity reached an optical density of 0.6 at 600 nm.</li>
 <li>Centrifuge 10 min at 4,000 rpm (4 &deg;C) and resuspend the pellet in 5 ml of 50 mM Tris buffer.</li>
 <li>Sonicate (Cell disrupter, LA Biosystems) the cells at 40% duty cycle with an output control of 4, keep the solution on ice for the entire duration.</li>
 <li>Centrifuge the resulting mixture for 5 min at 4,000 rpm at 4 &deg;C to remove the cell debris. Transfer the supernatant to a fresh vial.</li>
 <li>Determine the total protein concentration of the cell extracts by Bradford assay. Note: Use glass vials to prevent the increase of background and/or loss of protein.</li>
 <li>Prepare a 100 ml mixture containing 0.1% v/v alkane and 50 mM Tris-HCl buffer.</li>
 <li>Heat the mixture at 100 &deg;C for 5 min. Note: Perform this step only for medium-long chain alkanes with high boiling point. (e.g. C16: 287 &deg;C).</li>
 <li>To achieve an optimal solubility of the alkane, sonicate for 1 min while still warm until a homogenous, viscous mixture is obtained.</li>
 <li>Add 1mM of NADH, 1 mM FMN, 1 mM MgSO4 and 0.01 v/v Triton X-100.</li>
 <li>Prepare 6 ml aliquots in 25 ml closed-cap flasks.</li>
 <li>Add adequate amounts of cell extract (depends on Bradford assays, final concentration of 5 mg protein/l). Prepare a no-protein control.</li>
 <li>Incubate at 60 &deg;C (for optimal enzymatic activity) for 24 hr.</li>
 <li>Extract the hydrocarbons in the culture media by ethyl acetate and determine hydrocarbon concentration in cell culture by gas chromatography (see protocol 3).</li>
 <li>Calculate the degradation per unit of biomass by dividing the total amount of alkane converted by total protein added to each flask (by Bradford calibration curve). Further dividing by experiment duration yields the degradation activity per unit of cellular protein per time unit. The average is taken from three individual runs.</li>
</ol>
4. Ethyl Acetate Hydrocarbon Extraction and Concentration Measurements
<ol>
 <li>Alkanes are extracted from the aqueous solution by adding 2.5 ml ethyl acetate (apolar solvant) to 6 ml experimental solution. An internal standard is added to the solvent at a concentration of 0.1 % (v/v). The standard (e.g. cyclo-decan) varied depending on the expected range of the peaks of interest.</li>
 <li>Optional: Add Triton X-100 to the aqueous mixture and centrifuge the samples for 10 min at 4,000 rpm in order to get a proper bi-phasic system.</li>
 <li>Vortex the mixture for 5 sec (1,500 rpm) and incubate at room temperature until the two phases separate.</li>
 <li>Remove a maximal amount of the organic layer (top) and dry the solvent using anhydrous magnesium sulphate.</li>
 <li>Remove MgSO4 by filtration (0.2 &mu;m) and transfer the filtrate into gas chromatograph vials for measurements, or store at -20 &deg;C.</li>
 <li>Determine the concentration by gas chromatography using a CP-SIL 5CB column (length = 5 m). Inject 10 &mu;l of sample in split mode (1:10, 230 &deg;C). Set the column gas flow to 1 ml/min (Helium). The following oven temperature program is used:
 <table border="1">
 <tbody>
 <tr>
 <td>Rate</td>
 <td>Temperature [&deg;C]</td>
 <td>Time [min]</td>
 </tr>
 <tr>
 <td>0</td>
 <td>50</td>
 <td>7.5</td>
 </tr>
 <tr>
 <td>50</td>
 <td>90</td>
 <td>1.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>110</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>130</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>145</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>160</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>170</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>185</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>210</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>250</td>
 <td>2.0</td>
 </tr>
 <tr>
 <td>50</td>
 <td>320</td>
 <td>2.0</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>Integrate peaks and correct the concentrations with respect to the internal standard.</li>
</ol>
5. Alcohol/aldehyde Dehydrogenase Activity Assay
This assay was performed essentially according to the method described by Kato et al. (2010).
<ol>
 <li>Culture E. coli cells expressing the ADH (<a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a>) or ALDH (<a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a>) gene and cells carrying an empty vector (<a href="http://partsregistry.org/Part:BBa_J13002" target="_blank">BBa_J13002</a>) in 50 ml LB medium with appropriate antibiotics overnight.</li>
 <li>Transfer 500 &mu;l of the overnight culture into 50 ml of fresh LB (with antibiotic) and incubate until the cell turbidity reached an optical density of 0.6 at 600 nm.</li>
 <li>Centrifuge 10 min at 4,000 rpm at 4 &deg;C and resuspend the pellet in 5 ml of 50 mM Tris buffer.</li>
 <li>Sonicate the cell solution at 40% duty cycle with an output control of 4 (keep the solution on ice during the sonication).</li>
 <li>Centrifuge the resulting mixture for 5 min at 4,000 rpm at 4 &deg;C to remove cell debris.</li>
 <li>Determine the total protein concentration of the cell extracts by Bradford assay (Note: use a glass vial to prevent protein binding).</li>
 <li>Load a 96 well plate with 180 &mu;l 57 mM glycine buffer containing NAD (final concentration 1 mM, pH 9.5) in each well.</li>
 <li>Add 5 &mu;l of the alcohol (aldehyde) to be tested to the wells. Note: Heat long chain alcohols before the start of the assay to have a liquid. For each alcohol (aldehyde) a control without substrate should be added (negative control). In addition, prepare a blank containing a mixture of buffer and the substrate without cell extract.</li>
 <li>Preheat the plate of the plate reader (Tecan Magellan v7.0) for 15 min at 37 &deg;C to allow equilibration of the system.</li>
 <li>Add adequate amounts of cell extract (depends on Bradford assays, final concentration of 5 mg protein/l). Prepare a no-protein control.</li>
 <li>Measure the NADH production using a spectrophotometer at a wavelength of 340 nm every 2-3 min for 1 hr at 37 &deg;C.</li>
 <li>Calculate the NADH production rate from the slope of the OD (340 nm). Take into account the light path length and the extinction coefficient of NADH of 6220 M-1cm-1. Divide the NADH production rate by the total amount of protein added to express the activity of the dehydrogenase reaction in the cell extract (U/mg whole cell protein). Calculate the mean and the standard deviation from three independent runs.</li>
</ol>
6. pCaiF Characterization
<ol>
 <li>Culture E. coli cells expressing the pCaiF-GFP construct (<a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a>) and cells carrying the promoter alone (<a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326</a>) overnight in 5 ml LB medium the appropriate antibiotics overnight.</li>
 <li>Inoculate 5 ml M9 containing 10 g/l glucose and antibiotics with 50 &mu;l of the overnight culture and grow overnight.</li>
 <li>Subculture 50 &mu;l of the overnight outgrowth into 5 ml of fresh M9 with10 g/l and incubate until the cell turbidity reached an optical density of 0.2 at 600 nm.</li>
 <li>Load a 96 well plate with 100 &mu;l of fresh M9 medium containing the desired amount of carbon source for testing in each well. Perform triplicate experiments for statistical evaluation and add the respective negative controls (wild-type E. coli K12).</li>
 <li>Add 5 &mu;l of the overnight culture to the medium containing wells.</li>
 <li>Measure the growth curve (OD600) and GFP fluorescence (485 nm excitation and 520 emission) using a plate reader every 10 min for 18 hr at 37 &deg;C with constant shaking.</li>
 <li>Calculate the growth rate and the specific GFP content from the respective measurements and compare to the control. Calculate the mean and the standard deviation of at least three independent experiments.</li>
</ol>
7. Tolerance Assay
<ol>
 <li>Culture E. coli expressing PhPFD&alpha; and &beta; gene (<a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a>) overnight in 5 ml LB medium and the appropriate antibiotics. E. coli expressing ladA gene (<a href="http://partsregistry.org/Part:BBa_K398017" target="_blank">BBa_K398017</a>) is used as negative control.</li>
 <li>Subculture 10 &mu;l of the overnight outgrowth into fresh 5 ml of LB (with antibiotic) and incubate until the cell turbidity reached an optical density of 0.3 to 0.4 at 600 nm.</li>
 <li>Dilute the cultures with fresh medium until an OD600 of 0.1 is reached.</li>
 <li>Load a 96 well plate with 180 &mu;l M9 medium containing the appropriate antibiotics and the proper final concentration of the toxic compound (e.g. 0, 4, 8, 10% of n-hexane) in triplicate. Because alkane-water mixtures could lead to two-phase systems it is essential to have appropriate controls on the plate (e.g. different strains and the respective blank experiments).</li>
 <li>Add 20 &mu;l of the culture (control) into the wells.</li>
 <li>Measure the biomass concentration (OD600) using a plate reader every 10 min for 24 hr at 37 &deg;C with constant shaking.</li>
 <li>Calculate the growth rates from the respective measurements for the different agent concentrations and compare to the negative control. Calculate the mean and the standard deviation of at least three independent runs.</li>
</ol>
8. Homolog Interaction Mapping
<ol>
 <li>The application HIM was developed to perform protein queries on a PostgreSQL server running the STRING database (free for academic use) 20. The homologue interaction mapping application identifies interacting proteins in the original host organism using the STRING database. The sequences of the respective interacting genes are used in a BLAST search to find homologous genes in the target organism. Cytoscape 4 is used to visualize the result of the mapping. The HIM software tool can be downloaded at: <a href="https://github.com/jcnossen/InteractionHomologMapping" target="_blank">https://github.com/jcnossen/InteractionHomologMapping.</a></li>
 <li>To perform a mapping, (1) enter the BioBrick ID and the application will automatically download the part sequence data from the Registry of Standard Biological Parts 9, or (2) enter (paste) the sequence data in the application.</li>
 <li>Use the STRING Database website to find the STRING protein ID for the entered amino acid sequence.</li>
 <li>A protein with high homology is determined using BLAST. Subsequently the application lists each known interacting protein in the source organism and searches for homologs in the host organism (e.g. E. coli).</li>
 <li>Export resulting putative interaction list to text or Cytoscape.</li>
</ol>

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No conflicts of interest declared.

The BioBrick principle is used to construct a chassis for the degradation of alkanes and a proof of principle for the single components of the toolkit was obtained. Several assays are proposed to measure the in vivo and in vitro activity of alkane degrading pathway enzymes. The presented work successfully demonstrates a number of methods that can be used to determine enzyme activities and expression in the host organism E. coli after implementation of suitable BioBricks. Furthermore, it is show...

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 &beta;-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 phPFD&alpha; and phPFD&beta;, 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.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>E. coli K12</td>
			<td>New England Biolabs</td>
			<td>C2523H</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Octane</td>
			<td>Fluka</td>
			<td>74822</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexadecane</td>
			<td>Fluka</td>
			<td>52209</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>octanol-1</td>
			<td>Fluka</td>
			<td>95446</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>dodecanol-1</td>
			<td>Sigma-Aldrich</td>
			<td>126799</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>296090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Sigma</td>
			<td>N4505</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FMN</td>
			<td>Sigma</td>
			<td>F2253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>J.T. Baker Casno</td>
			<td>7487 889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell disrupter</td>
			<td>LA Biosystems</td>
			<td>CD-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Amersham pharmacia</td>
			<td>spec 2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Tecan</td>
			<td>Magellan v7.0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Innova, 44</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BioBrick K398014: 
			BBa_J23100-BBa_J61100-alkB2-BBa_J61100-rubA3-BBa_J61100-rubA4- BBa_J61100-rubB</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_K398014</a></td>
			<td>Alkane Hydroxylase System 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398027: BBa_R0040-BBa_B0034-ladA</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398027" target="_blank">BBa_K398027</a></td>
			<td>ladA Protein Generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398018: BBa_J23100-BBa_J61101-ADH</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398018" target="_blank">BBa_K398018</a></td>
			<td>ADH generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398030: BBa_R0040-BBa_B0034-ALDH</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398030" target="_blank">BBa_K398030</a></td>
			<td>ALDH generator 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398326: pCaiF</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398326" target="_blank">BBa_K398326</a></td>
			<td>pCaiF promoter 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398331: pCaiF-BBa_B0032-BBa_I13401</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398331" target="_blank">BBa_K398331</a></td>
			<td>pCaiF measurement device 
			Resistance: Chloramphenicol</td>
		</tr>
		<tr>
			<td>BioBrick K398406: BBa_J23002-BBa_J61107-phPFD&alpha;-BBa_J61107-</td>
			<td>Delft University of Technology at the department of Biotechnology or Registry of Standard Biological Parts</td>
			<td><a href="http://partsregistry.org/Part:BBa_K398406" target="_blank">BBa_K398406</a></td>
			<td>Solvent tolerance cluster 
			Resistance: Chloramphenicol</td>
		</tr>
	</tbody>
</table>

a toolkit to enable hydrocarbon conversion in aqueous environments

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 &beta;-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, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; 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.

Alkane conversion
The activity of the three oxidation steps from the alkane to the respective fatty acid was evaluated using resting cell assays and enzyme activity measurements. The results are presented following the pathway reactions (1) alkane hydroxylase, (2) alcohol dehydrogenase and (3) aldehyde dehydrogenase. 
For the first step, different plasmids were constructed for medium and long-chain alkanes. The plasmid <a href="http://partsregistry.org/Part:BBa_K398014" target="_blank">BBa_...

Watch this Scientific Journal Video about A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments at JoVE.com

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

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 ...

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SCIENTISTS IN ACTION

14.0K 조회수.  Delft University of Technology. 오일 오염의 개선을 위해 박테리아 시스템을 규제 지속 가능한 자동차는 표준 호환 DNA 부품 (BioBricks)를 사용하여 설계되었습니다. 엔지니어링 E. 대장균 변형은 독성 수성 환경에서 β-산화를 통해 alkanes을 저하하는 데 사용되었다. 종에서 각각의 효소는 alkane 저하 활동을 보여 주었다. 에 또한 증가 허용 N - 헥산은 alkane에 강한 박테리아 ?...

비디오: A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...