http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Ptmvanboheemen&year=&month=2010.igem.org - User contributions [en]2024-03-28T08:12:26ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:TU_Delft/EPACTeam:TU Delft/EPAC2010-10-27T23:16:21Z<p>Ptmvanboheemen: </p>
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<div>__NOTOC__<br />
==The EPAC Pyramid==<br />
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===Introduction===<br />
<br />
When new technologies are presented to society, responses of the public are hard to predict. In the case of synthetic biology numerous questions rise about safety, security and ethics. Also issues like patenting versus open-source models are a topic of debate. During our iGEM project we tried to analyze, assess and improve the relationship between society and synthetic biology. <br />
With our new approach, our team hopes to improve the way in which synthetic biology meets the expectations and needs of society. <br />
<br />
===Inspiration===<br />
<br />
For our Human Practice project we got inspired by a 2-year European Project called SYNBIOSAFE (1). <br />
SYNBIOSAFE was the first project in Europe to research the safety and ethical aspects of synthetic biology, aiming to proactively stimulate a debate on the following issues: safety, security, ethics and the boundary between science and society (2,3).<br />
<br />
Based on this project, we developed a complete new approach to human practice: The EPAC pyramid. Our model consists out of four elements: '''E'''ducation, '''P'''erception, '''A'''wareness & '''C'''ollaboration. These fundamentals formed the basis for organizing our activities. Because these elements are strongly related to each other we linked them together: the pyramid was born. <br />
<br />
[[Image:TU Delft Flowchart for EPAC.jpg|400px|center|thumb|Figure 1. Depending on the general public opinion and awareness, scientist can provide the right information to inform about science & technology. People recieve this information and process this according to their own values and standards. After processing this information, people will be better able to join the discussion on science & technology.]]<br />
<br />
===EPAC - This is how it works===<br />
<br />
Using our unique and holistic EPAC approach, we could really focus on the different disciplinaries involving human practice and help society to consider, guide and address the impacts of ongoing advances in synthetic biology.<br />
Working with the model makes it important to first distinguish two information streams. Information can be received and distributed (figure 1).<br />
<br />
In our point of view, we are at the interface between science and society. We can receive from and spread information to both sides. <br />
By providing information we aim to make people '''aware''' or more '''educated''' in the field of synthetic biology.<br />
The direct consequence of processing this information leads to the formation of '''perception'''. These opinions are not only based on the newly absorbed information, but also on norms, values, upbringing and education of the recipient. <br />
<br />
Therefore it is imposible to look at education, perception and awareness as separate element. There is a continuous exchange between these streams of communication.<br />
We used the principle of the adressing two of this three elements in all of our activities and thereby maintaining the connection between the elements.<br />
<br />
The C in EPAC comes from [[Team:TU_Delft/Collaboration|collaboration]]. Without cooperation, all of the above is not possible.<br />
<br />
<br />
''With the EPAC pyramid we provided a standard model for everyone that aims to manage their activities in the ongoing debate in the field of synthetic biology.''<br />
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===Our activities in the EPAC===<br />
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Click on the icons on the EPAC-pyramid to check our human practice activities.<br />
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===References===<br />
#'''Ganguli-Mitra, A., M. Schmidt, et al.''' (2009). Of Newtons and heretics. ''Nat Biotechnol'' 27(4): 321-322.<br />
#'''Schmidt, M., A. Ganguli-Mitra, et al.''' (2009). A priority paper for the societal and ethical aspects of synthetic biology. ''Syst Synth Biol'' 3(1-4): 3-7.<br />
#'''Schmidt, M., H. Torgersen, et al.''' (2008). SYNBIOSAFE e-conference: online community discussion on the societal aspects of synthetic biology. ''Syst Synth Biol'' 2(1-2): 7-17.</div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/conclusionsTeam:TU Delft/Project/conclusions2010-10-27T23:05:51Z<p>Ptmvanboheemen: /* Sub-projects */</p>
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<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Conclusions==<br />
We have shown that using the concept of BioBricks it is possible to design an organism that ('''1''') reacts to its environment [[Team:TU_Delft/Project/sensing|(sensing)]], ('''2''') influences the solubility of hydrocarbons [[Team:TU_Delft/Project/solubility|(solubility)]] , ('''3''') exhibits a higher tolerance towards solvents and salts [[Team:TU_Delft/Project/tolerance|(survival)]] and ('''4''') implements (parts) of novel pathways [[Team:TU_Delft/Project/alkane-degradation|(alkane degradation)]]. This chassis could be used for example the biological degradation of residual oil in oil sands tailing waters, or the treatment of waste water from the oil industry. <br />
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[[Image:TUDelft_Group.png|center]]<br />
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==Sub-projects==<br />
<html><div style="float:right"><a href="https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation"><img src="https://static.igem.org/mediawiki/2010/2/21/TU_Delft_degradation_icon.jpg" align="right" vspace="5" hspace="5" border="0" /></div></a><br />
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<h3><a href="https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation" style="color: #9f0f0e;"><br />
Alkane degradation<br />
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According to our analysis, the enzymatic activities of our recombinant strain and the negative control are statistically different at confidence level of 0.95, which means that the part BBa_K398018 the alcohol dehydrogenase activity by a factor two. We showed that the parts BBa_K398005 and BBa_K398018 have biological activity; particularly when we used BBa_K398018 the enzyme activity of E. coli cell extracts became equivalent to 3% of the in vitro activity of the positive control (''Pseudomonas putida'' OTR1). <br />
<br />
Our results also suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination BBa_J23100-BBa_J61117 increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part BBa_J13002 as promoter-rbs combo increases the same activity 3-fold. These enzymatic activities are equivalent to 33.98% and 42.01% of the Pseudomonas putida aldehyde dehydrogenase activity, respectively.<br />
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<html><div style="float:right"><a href="https://2010.igem.org/Team:TU_Delft/Project/sensing"><img src="https://static.igem.org/mediawiki/2010/b/bd/TU_Delft_Sensing_icon.jpg" align="right" vspace="5" hspace="5" border="0" /></a></div><br />
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<h3><a href="https://2010.igem.org/Team:TU_Delft/Project/sensing" style="color: #6aab2c;"><br />
Sensing<br />
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After the pre cultures have been grown in glucose, a switch to alkanes as a sole cabon and energy source is required. Therefore we designed a biobrick switch that can sense the absence of glucose: pCaif.<br />
<br />
This new promoter combined with B0032 has a GFP production rate of 3.975E07 GFP molecules/second/O.D. during the stationary phase. Moreover, we demonstrated that this promoter is more active the during stationary phase indicating high cAMP levels. Finally, in the presence of a secondary carbon source the GFP production rate decreases again due to the catabolism of the secondary C-source.<br />
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<html><div style="float:right"><a href="https://2010.igem.org/Team:TU_Delft/Project/tolerance"><img src="https://static.igem.org/mediawiki/2010/5/59/TU_Delft_Tolerance_icon.jpg" align="right" vspace="5" hspace="5" border="0" /></div></a><br />
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<h3><a href="https://2010.igem.org/Team:TU_Delft/Project/tolerance" style="color: #002f82;"><br />
Survival<br />
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====Salt tolerance====<br />
Our biobrick has enabled us to increase the salt tolerance of E.coli by an average of 20%. The presented BioBrick probably reduces one of several effects caused by salt stress. A complete tolerance using a single protein seems unrealistic, a combination of different could further increase the tolerance to salt.<br />
<br />
====Solvent tolerance====<br />
Our BioBrick has enabled us to increase the solvent tolerance of ''E.coli'' when n-hexane is present in the culture medium at high concentrations. According to our findings, the growth rate of ''E.coli'' is improved 60% at a n-hexane concentration of 10%(v/v).<br />
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<html><div style="float:right"><a href="https://2010.igem.org/Team:TU_Delft/Project/solubility"><img src="https://static.igem.org/mediawiki/2010/9/98/TU_Delft_Solubility_icon.jpg" align="right" vspace="5" hspace="5" border="0" /></div></a><br />
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Solubility<br />
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To overcome the mass‐transfer limitation between the water and oil phase, a gene encoding for [[Team:TU_Delft/Project/solubility/parts|AlnA]], a protein with emulsifying properties was expressed. The [[Team:TU_Delft/Project/solubility/results|increased solubility]] is about 20%. The solubility was determined by [[Team:TU_Delft/Project/solubility/characterization|a new method]]. We suggest that in future research the protein is tagged, so it can be isolated with higher purity.<br />
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<html><div style="float:right"><a href="https://2010.igem.org/Team:TU_Delft/Project/rbs-characterization"><img src="https://static.igem.org/mediawiki/2010/0/06/TU_Delft_RBS_characterization_icon.jpg" align="right" vspace="5" hspace="5" border="0" /></div></a><br />
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RBS Characterization<br />
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In order to fine-tune the protein expressions of the alkane degrading genes we've [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization characterized] 5 members of the Anderson RBS family using an improved protein production model taking dilution and protein degradation into account. The following relative efficiencies were found:<br />
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>RBS</b><br />
|<b>Efficiency</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|1.20%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|11.9%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|7.70%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|1.26%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|6.52%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|30.0%<br />
|}<br />
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<html></div><br style="clear:both;" /></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/referencesTeam:TU Delft/Project/references2010-10-27T22:59:12Z<p>Ptmvanboheemen: </p>
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<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==References==<br />
<br />
====Alkane Degradation====<br />
#'''Fujii, T., Narikawa, T., Takeda, K., Kato, J.''', Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from ''Gordonia sp. TF6''. ''Bioscience, biotechnology, and biochemistry'', 68(10) 2171-2177 ('''2004''')<br />
#'''Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao.''' Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. ''Journal of molecular biology'', 376: 453-465 ('''2008''')<br />
#'''Tomohisa Kato, Asuka Miyanaga, Mitsuru Haruki, Tadayuki Imanaka, Masaaki Morikawa & Shigenori Kanaya.''' Gene Cloning of an Alcohol Dehydrogenase from Thermophilic Alkane-Degrading ''Bacillus thermoleovorans B23''. ''Journal of Bioscience and Bioengineering'' 91(1):100-102 ('''2001''')<br />
#'''Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa.''' Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans B23''. ''Extremophiles'' 14:33-39 ('''2010''')<br />
#'''Sulzenbacher, G., et al.''', Crystal structure of E-coli alcohol dehydrogenase YqhD: Evidence of a covalently modified NADP coenzyme. ''Journal of Molecular Biology'' 342(2):489-502 ('''2004''')<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#'''Hoffmann F. and Rinas U'''. Stress Induced by Recombinant Protein Production in ''Escherichia coli'' ''Advances in Biochemical Engineering/Biotechnology'', Vol. 89, pp. 73-92.('''2004''')<br />
<br />
====Sensing====<br />
#'''Canosa, I., J. M. Sanchez-Romero, et al.''' A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. ''Molecular Microbiology'' 35(4): 791-799 ('''2000''')<br />
#'''Moreno, R., A. Ruiz-Manzano, et al.''' The Pseudomonas putida Crc global regulator is an RNA binding protein that inhibits translation of the AlkS transcriptional regulator. ''Molecular Microbiology'' 64(3): 665-675 ('''2007''')<br />
#'''van Beilen, J. B., S. Panke, et al.''' Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes. ''Microbiology-Sgm'' 147: 1621-1630 ('''2001''')<br />
#'''Rojo, F.''' , Degradation of alkanes by bacteria. ''Environmental Microbiology'' 11: 2477-2490 ('''2009''')<br />
#'''Kotte, O, Zaugg, J., Heinemann, M. ''', ‘Bacterial adaptation through distributed sensing of metabolic fluxes’, ''Molecular Systems Biology'', 6:355, doi:10.1038/msb.2010.10 ('''2010''')<br />
#'''Kremling, A., Bettenbrock, K., Gilles, E.D.''', ‘Analysis of global control of Escherichia coli carbohydrate uptake’, ''BMC Systems Biology'', 1:42, doi:10.1186/1752-0509-1-42 ('''2007''')<br />
#'''Lin, H. Y., Mathiszik, B., Xu, B., Enfors, S.-O., Neubauer, P.''', ‘Determination of the Maximum Specific Uptake Capacities for Glucose and Oxygen in Glucose-Limited Fed-Batch Cultivations of ''Escherichia coli''’, ''Biotechnology and Bioengineering'', 73, 347-357 ('''2001''')<br />
#'''Alon, U. (ed.)''', An Introduction to Systems Biology: Design Principles of Biological Circuits, ''CRC Press'' ('''2007''')<br />
#EcoCyc: [http://BioCyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=CPLX0-226 click here]<br />
<br />
====Survival====<br />
#'''S. Tanaka,K. Ikeda, H. Miyasaka''', Enhanced Tolerance Against Salt-Stress and Freezing-Stress of Escherichia coli Cells Expressing Algal bbc1 Gene. ''Current Microbiology'', 42:173-177 ('''2001''')<br />
#'''Y. Suda,T. Yoshikawa,Y. Okuda,M. Tsunemoto,S. Tanaka,K. Ikeda,H. Miyasaka,M. Watanabe,K. Sasaki,K. Harada,T. Bamba,K. Hirata''', Isolation and characterization of a novel antistress gene from ''Chlamydomonas sp. W80''. ''Journal of Bioscience and Bioengineering'', 107(4) 352-354 ('''2009''')<br />
#'''Y. Hase, S. Yokoyama, A. Muto, et al.''' , Removal of a ribosome small subunit-dependent GTPase confers salt resistance on Escherichia coli cells. ''RNA Society'', 15:1766-1774 ('''2009''')<br />
#'''Mihaela Marilena Lăzăroaie''',Investigation of saturated and aromatichydrocarbon resistance mechanismsin ''Pseudomonas aeruginosa'' IBB ''Cent. Eur. J. Biol.'' 4(4) 469-481 ('''2009''')<br />
#'''M. Okochi, K. Kanie, M. Kurimoto ,M. Yohda & Hiroyuki Honda''' Overexpression of prefoldin from the hyperthermophilic archaeum ''Pyrococcus horikoshii'' OT3 endowed ''Escherichia coli'' with organic solvent tolerance ''Appl Microbiol Biotechnol'' 79:443-449 ('''2008''')<br />
====Solubility====<br />
#'''Walzer, G., Rosenberg, E. and Ron, E.Z.''' The Acinetobacter outer membrane protein A (OmpA) is a secreted emulsifier. ''Environmental Microbiology.'' 8:1026-1032.('''2006''')<br />
#'''Toren, A., Segal, G., Ron, E.Z. and Rosenberg, E.''' Structure--function studies of the recombinant protein bioemulsifier AlnA. ''Environmental Microbiology.'' 4:257-261.('''2002''')<br />
#'''Navon-Venezia, S., et al.''' Alasan, a new bioemulsifier from Acinetobacter radioresistens. ''Applied and Environmental Microbiology.'' 61:3240-3244.('''1995''')<br />
#'''Toren, A., Orr, E., Paitan, Y., Ron, E.Z. and Rosenberg, E.''' The active component of the bioemulsifier alasan from Acinetobacter radioresistens KA53 is an OmpA-like protein. ''The Journal of Bacteriology.'' 184:165-170.('''2002''')<br />
#'''Toren, A., Navon-Venezia, S., Ron, E.Z. and Rosenberg, E.''' Emulsifying activities of purified Alasan proteins from Acinetobacter radioresistens KA53. ''Applied and Environmental Microbiology.'' 67:1102-1106.<br />
#'''Ron, E.Z. and Rosenberg, E.''' (2002) Biosurfactants and oil bioremediation. ''Current Opinion in Biotechnology.'' 13:249-252.('''2001''')<br />
#'''Suresh Kumar, A., Mody, K. and Jha, B.''' Evaluation of biosurfactant/bioemulsifier production by a marine bacterium. ''Bulletin of Environmental Contamination and Toxicology.'' 79:617-621.('''2007''')</div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Team:TU Delft/2010-10-27T22:33:37Z<p>Ptmvanboheemen: Redirecting to Team:TU Delft</p>
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<div>#REDIRECT [[Team:TU_Delft]]</div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_DelftTeam:TU Delft2010-10-27T22:23:33Z<p>Ptmvanboheemen: </p>
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<br />
// Set document ready callback<br />
$(function() {<br />
if (wgUserName) {<br />
// Create content edit link<br />
$(".left-menu ul > *:last").after('<li><a id="edit_content_link">edit content</a></li>');<br />
updateContentEditLink();<br />
}<br />
<br />
// Initialize history plugin.<br />
$(window).hashchange(historyCallback);<br />
rewriteLinks($("#navlist"));<br />
<br />
historyCallback();<br />
<br />
// setup hovering<br />
$(".menu_button").hover(<br />
function () { $(this).addClass("hover"); },<br />
function () { $(this).removeClass("hover"); }<br />
);<br />
});<br />
<br />
<br />
function isDefined(variable)<br />
{<br />
return typeof(window[variable]) != "undefined";<br />
}<br />
<br />
function splitHash(hash) {<br />
var kvpairs = hash.split(ampersandStr);<br />
var i;<br />
var kvmap = {};<br />
for(i=0;i<kvpairs.length;i++) {<br />
var s = kvpairs[i].split('=');<br />
kvmap[s[0]] = s[1];<br />
}<br />
return kvmap;<br />
}<br />
<br />
function makeHash(page, kvmap) {<br />
var str = '#page=' + page;<br />
var i;<br />
if (kvmap) {<br />
for(i in kvmap) {<br />
str += ampersandStr + i + '=' + kvmap[i];<br />
}<br />
}<br />
return str; <br />
}<br />
<br />
function setHash(page, kvmap) {<br />
location.hash = makeHash(page,kvmap);<br />
}<br />
<br />
<br />
function include_js(file, cb) {<br />
var html_doc = document.getElementsByTagName('head')[0];<br />
var js = document.createElement('script');<br />
js.setAttribute('type', 'text/javascript');<br />
js.setAttribute('src', file);<br />
html_doc.appendChild(js);<br />
<br />
js.onreadystatechange = function () {<br />
if (js.readyState == 'complete')<br />
cb();<br />
}<br />
<br />
js.onload = cb;<br />
}<br />
<br />
function loadScript(src, callback)<br />
{<br />
include_js(src, callback);<br />
}<br />
<br />
<br />
function moveToAnchor(anchor) {<br />
try {<br />
var pos = $('#iGEM_TU_Delft_container > a[name='+anchor+']').offset();<br />
dbgout('anchor: '+anchor+'; pos=' + pos.left + ','+pos.top );<br />
window.scroll(pos.left, pos.top);<br />
} catch (err) {<br />
dbgout('moveToAnchor error: ' + err);<br />
}<br />
}<br />
<br />
<br />
function loadPage(page, anchor)<br />
{<br />
var showLoadAnim = !!currentPage;<br />
currentPage = page;<br />
var url = wgServer + "/Team:TU_Delft/" + page + "?action=render";<br />
if(page.substring(0, 5) == "User:") {<br />
url = wgServer + "/" + page + "?action=render";<br />
}<br />
$(window).trigger('page_close');<br />
<br />
if(showLoadAnim) {<br />
$("#TUD-loading-panel").show();<br />
//$("#loading-overlay").show();<br />
$("#iGEM_TU_Delft_container").fadeTo(200,0.25);<br />
$('html, body').animate({scrollTop:0}, 'slow');<br />
}<br />
loading_home = false;<br />
<br />
var processPage = function(next) {<br />
dbgout('processPage: ' + page);<br />
$(window).trigger('page_init');<br />
rewriteLinks($("#iGEM_TU_Delft_container"));<br />
updateContentEditLink();<br />
$("#iGEM_TU_Delft_container").fadeTo(200,1).delay(200).queue(function(n) {<br />
if(anchor) moveToAnchor(anchor); n();<br />
});<br />
next();<br />
}<br />
<br />
$.get(url, function(data) {<br />
$("#iGEM_TU_Delft_container").html(data).queue(processPage);<br />
buildBreadCrumbTrail(page); <br />
Cufon.replace('h2'); // Works without a selector engine <br />
Cufon.replace('h3'); // Works without a selector engine <br />
Cufon.replace('#sub1'); // Requires a selector engine for IE 6-7, see above <br />
$("tr:nth-child(odd)").addClass("odd");<br />
<br />
$("#TUD-loading-panel").hide();<br />
$("#loading-overlay").hide();<br />
});<br />
}<br />
<br />
function historyCallback() {<br />
var hash = location.hash;<br />
if(hash) {<br />
hash = hash.substring(1);<br />
if(hash.split('=').length > 1) {<br />
var kvmap = splitHash(hash);<br />
var changepage;<br />
<br />
// looks a little clumsy, but js AND operator conflicts with mediawiki markup<br />
if (kvmap.page) if(kvmap.page != currentPage) changepage = kvmap.page;<br />
<br />
if (changepage) {<br />
loadPage(changepage, kvmap.anchor);<br />
} else {<br />
if(kvmap.anchor) moveToAnchor(kvmap.anchor);<br />
$(window).trigger('hashupdate');<br />
}<br />
}<br />
} else {<br />
setHash('Home');<br />
}<br />
}<br />
<br />
function rewriteLinks(elem) {<br />
$("a",elem).each(function() {<br />
var txt = $(this).text();<br />
var url = this.href;<br />
<br />
if(this.hash) {<br />
var anchor = this.hash.substring(1);<br />
<br />
if (anchor.substring(0,5)!='page=')<br />
this.href = '#page=' + currentPage + ampersandStr + 'anchor=' + anchor;<br />
} else if(txt != "edit")<br />
this.href = this.href.replace("https://2010.igem.org/Team:TU_Delft/", "#page=");<br />
<br />
// dbgout('rewriting ' + url + ' to ' + this.href);<br />
});<br />
}<br />
<br />
<br />
</script><br />
<script type="text/javascript"><br />
var timeout = 500;<br />
var closetimer = 0;<br />
var ddmenuitem = 0;<br />
<br />
function jsddm_open() {<br />
jsddm_canceltimer();<br />
jsddm_close();<br />
ddmenuitem = $(this).find('ul').css('display', 'block');<br />
//ddmenuitem = $(this).find('ul').show(200);<br />
}<br />
<br />
function jsddm_close() {<br />
if(ddmenuitem) ddmenuitem.css('display', 'none');<br />
//if(ddmenuitem) ddmenuitem.hide();<br />
}<br />
<br />
function jsddm_timer() {<br />
closetimer = window.setTimeout(jsddm_close, timeout);<br />
}<br />
<br />
function jsddm_canceltimer() {<br />
if(closetimer) {<br />
window.clearTimeout(closetimer);<br />
closetimer = null;<br />
}<br />
}<br />
<br />
$(function() {<br />
$('#navlist > li').bind('mouseover', jsddm_open);<br />
$('#navlist > li').bind('mouseout', jsddm_timer);<br />
// $("#navlist li ul li:even").addClass("alt");<br />
});<br />
<br />
document.onclick = jsddm_close;<br />
</script><br />
<br />
<script language="javascript" type="text/javascript"><br />
function capitaliseFirstLetter(string)<br />
{<br />
return string.charAt(0).toUpperCase() + string.slice(1);<br />
}<br />
<br />
function buildDepth(array,count)<br />
{<br />
var depthStr="";<br />
for (i=0;i<count;i++)<br />
{<br />
depthStr=depthStr + array[i] + "/" ;<br />
}<br />
return depthStr;<br />
}<br />
function buildBreadCrumbTrail(page)<br />
{<br />
var constituentFolders = new Array();<br />
var currentURL = page;<br />
constituentFolders=currentURL.split("/");<br />
var outputStr="<a href='https://2010.igem.org/Team:TU_Delft#page=Home'>Home</a>";<br />
if(page != "Home") {<br />
for (count=0;count<(constituentFolders.length);count++)<br />
{<br />
if(constituentFolders[count].substring(0, 5) == "User:") {<br />
outputStr = outputStr + " <span class='doubleright'>&raquo;</span> <a href='https://2010.igem.org/Team:TU_Delft#page=Team/'>Team</a> <span class='doubleright'>&raquo;</span> <a href='https://2010.igem.org/Team:TU_Delft#page=Team/members'>Members</a> <span class='doubleright'>&raquo;</span> <a href='https://2010.igem.org/Team:TU_Delft#page=" + buildDepth(constituentFolders,count) + constituentFolders[count] + "'>" + capitaliseFirstLetter(constituentFolders[count]).replace(/-/gi," ").substring(5) + "</a>";<br />
}<br />
if(constituentFolders[count].substring(0, 5) !== "User:") {<br />
outputStr=outputStr + " <span class='doubleright'>&raquo;</span> <a href='https://2010.igem.org/Team:TU_Delft#page=" + buildDepth(constituentFolders,count) + constituentFolders[count] + "'>" + capitaliseFirstLetter(constituentFolders[count]).replace(/-/gi," ") + "</a>";<br />
}<br />
}<br />
}<br />
$("#breadcrumbs").html(outputStr);<br />
}<br />
</script><br />
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<br />
<!-- Main CSS - START /--><br />
<style type="text/css"><br />
/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0; width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#content { z-index: 1; background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; margin-top: -15px !important; margin-top: 0px;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#top-section { z-index: 2; height: 15px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; padding:0; border: none; font-size: 10px;}<br />
#p-logo { height:1px; overflow:hidden; display: none;}<br />
#search-controls { overflow:hidden; display:block; background: none; position: absolute; top: 100px; right: 40px;}<br />
.left-menu { width: 500px !important; display:block; margin-top:-80px; border: none; text-align: right;}<br />
.left-menu ul { border: none; }<br />
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}<br />
.right-menu ul { border: none; width: 300px;}<br />
#footer-box { background-color: #216085; border: none; width: 100%; margin: -10px auto 0 auto; padding: 20px 0;}<br />
.visualClear { display: none; }<br />
#footer { border: none; width: 965px; margin: 0 auto; padding: 0;}<br />
.firstHeading { display: none;}<br />
#f-list a { color: #333; font-size: 10px;}<br />
#f-list a:hover { color: #666;}<br />
.printfooter { display: none; }<br />
#footer ul { margin: 0; padding: 0;}<br />
#footer ul li { margin-top: 0; margin-bottom: 0; margin-left: 10px; margin-right: 10px; padding: 0;}<br />
#search-controls { display:none; }<br />
h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
p:first-child { display: none;}<br />
h1{border:none; width: 100%; clear: both;}<br />
/* Wiki Hacks - END */<br />
<br />
table {<br />
margin: 0;<br />
padding: 0;<br />
font-size: 11px;<br />
border: 1px solid #000;<br />
}<br />
table td {<br />
padding: 5px;<br />
}<br />
table .head {<br />
background-color: #216085 ;<br />
font-weight: bold;<br />
color: #fff;<br />
}<br />
table .odd {<br />
background-color: #d0f1fa;<br />
}<br />
<br />
h2 {<br />
font-size: 30px;<br />
border: none;<br />
}<br />
<br />
h3 {<br />
font-size: 20px;<br />
border: none;<br />
}<br />
<br />
html, body {<br />
margin: 0;<br />
padding: 0;<br />
width: 100%;<br />
height: 100%; <br />
}<br />
<br />
body {<br />
background-color: #d0f1fa;<br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
min-width: 1050px;<br />
}<br />
<br />
#TUD-main-wrapper {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/b/b3/TU_Delft_footer_tile.gif');<br />
background-position: bottom left;<br />
background-repeat: repeat-x; <br />
display: block; <br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
}<br />
<br />
#TUD-main-wrapper div {<br />
line-height: 18px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-main-wrapper2 {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/9/9d/TU_Delft_bg_tile.gif');<br />
background-repeat: repeat-x;<br />
background-repeat: repeat-x; <br />
display: block; <br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
margin-top: -8px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper2 {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-content-wrapper {<br />
background-color: #fff;<br />
text-align: left;<br />
width: 1024px;<br />
margin: 0 auto;<br />
padding: 0;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
<br />
#TUD-header {<br />
width: 1024px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/58/TU_Delft_header_bg.gif');<br />
height: 120px;<br />
}<br />
<br />
#TUD-menu {<br />
width: 1024px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/c/c5/TU_Delft_menu_bg.gif');<br />
height: 38px;<br />
}<br />
<br />
<br />
#TUD-body-wrapper {<br />
width: 100%;<br />
background-image: url('https://static.igem.org/mediawiki/2010/8/80/TU_Delft_body_bg.jpg');<br />
/* FireFox collapsing margin fix */<br />
padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
}<br />
<br />
#TUD-body-container {<br />
width: 100%;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5d/TU_Delft_body_top_bg.jpg');<br />
background-repeat: no-repeat;<br />
/* FireFox collapsing margin fix */<br />
padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
display: block;<br />
}<br />
<br />
#TUD-body-content {<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5f/TU_Delft_body_footer_bg.jpg');<br />
width: 1024px;<br />
background-repeat: no-repeat;<br />
background-position: bottom left;<br />
padding-bottom: 15px<br />
}<br />
<br />
#TUD-main-content {<br />
width: 904px;<br />
margin: 10px 60px 0px 60px;<br />
}<br />
<br />
#iGEM_TU_Delft_container {<br />
min-height: 400px;<br />
}<br />
<br />
#TUD-footer {<br />
width: 100%;<br />
height: 254px;<br />
text-align: center;<br />
}<br />
<br />
h1 {<br />
margin: 0;<br />
padding: 0;<br />
display: block;<br />
float: left;<br />
}<br />
<br />
.TUD-logo {<br />
background-image: url('https://static.igem.org/mediawiki/2010/0/09/TU_Delft_iGEM_Team_Logo.png');<br />
width: 237px;<br />
height: 95px;<br />
margin-left: 90px;<br />
background-repeat: no-repeat;<br />
border: none;<br />
}<br />
<br />
.TUD-logo span {<br />
visibility: hidden;<br />
}<br />
<br />
<br />
#TUD-loading-panel {<br />
z-index: 100;<br />
position: absolute;<br />
left: 0px;<br />
top: 0px;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
}<br />
<br />
#TUD-loading-panel-logo {<br />
margin: 0 auto;<br />
margin-top: 250px;<br />
}<br />
<br />
#loading-overlay {<br />
z-index: 70;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
opacity:0.4;<br />
filter:alpha(opacity=40);<br />
}<br />
<br />
#back_to_igem {<br />
margin: 15px 0 0 0;<br />
padding:0;<br />
float: right;<br />
}<br />
<br />
/* MENU */<br />
#navlist {<br />
margin: 0 0 0 55px;<br />
padding: 0;<br />
}<br />
<br />
#navlist li {<br />
display: block;<br />
list-style-type: none;<br />
float:left;<br />
padding: 0;<br />
margin: 0;<br />
}<br />
<br />
#navlist li a {<br />
text-decoration: none;<br />
color: #000;<br />
}<br />
<br />
#navlist li a:hover {<br />
text-decoration: underline;<br />
}<br />
<br />
#navlist li ul {<br />
position: absolute;<br />
display:none;<br />
background-color: #fff;<br />
padding:0;<br />
border: 1px solid #aaa;<br />
z-index:100;<br />
margin-left: 0px;<br />
margin-top: 4px;<br />
}<br />
<br />
html>body #navlist li ul {<br />
margin-left: 0;<br />
}<br />
<br />
#navlist li ul li {<br />
list-style-type:none;<br />
float:left;<br />
clear:both;<br />
z-index:100;<br />
}<br />
<br />
#navlist li ul li a {<br />
display: block;<br />
color: #222;<br />
width: 150px;<br />
border: 1px solid #d0f1fa;<br />
padding: 3px;<br />
}<br />
<br />
#navlist li ul li a:hover {<br />
color: #000;<br />
background-color: #54c9f5;<br />
text-decoration: underline;<br />
}<br />
<br />
#insilico_menu li a {<br />
width: 230px !important;<br />
}<br />
<br />
.menubutton {<br />
display:block;<br />
margin: 13px 10px 0px 10px;<br />
}<br />
/* FOOTER */<br />
#TUD-footer, #TUD-footer-content {<br />
width: 904px;<br />
margin: 0 auto;<br />
text-align: left;<br />
}<br />
/*TABLE .odd {<br />
background-color: #dbeaff;<br />
} */<br />
<br />
</style><br />
<!-- Main CSS - END /--><br />
<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
<!-- yes im using center --><br />
<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
</div><br />
</div><br />
<br />
<div id="TUD-main-wrapper2"><br />
<div id="TUD-main-wrapper"><br />
<br />
<div id="TUD-content-wrapper"><br />
<div id="TUD-header"><br />
<style><br />
a.nohover:hover {<br />
text-decoration: none;<br />
}<br />
</style><br />
<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
</div><br />
<div id="TUD-menu"><br />
<br />
<ul id="navlist"><br />
<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/statistics" title="Statistics">Statistics</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
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<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/HC_regulation" title="Hydrocarbon Regulation">Hydrocarbon Regulation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/protein-production-model" title="Protein production model">Protein production model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model" title="pCaiF CRP sensing model">pCaiF CRP sensing model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/interaction-mapping" title="Software Tool">Interaction Mapping</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Software/part-search" title="Part search server & iPhone app">Part search server & iPhone app</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks" title="Wiki tips & tricks">Wiki Tips &amp; Tricks</a></li><br />
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<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/in-the-news" title="In The News">In the Media</a></li><br />
<!-- <li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/articles" title="Articles">Articles</a></li> /--><br />
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<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/acknowledgements" title="Acknowledgements">Acknowledgements</a></li><br />
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</html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Tour/curtainTeam:TU Delft/Tour/curtain2010-10-27T22:21:37Z<p>Ptmvanboheemen: </p>
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</html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/EPACTeam:TU Delft/EPAC2010-10-27T21:55:57Z<p>Ptmvanboheemen: /* Our activities in the EPAC */</p>
<hr />
<div>__NOTOC__<br />
==The EPAC Pyramid==<br />
<br />
===Introduction===<br />
<br />
When new technologies are presented to society, responses of the public are hard to predict. In the case of synthetic biology numerous questions rise about safety, security and ethics. Also issues like patenting versus open-source models are a topic of debate. During our iGEM project we tried to analyze, assess and improve the relationship between society and synthetic biology. <br />
With our new approach, our team hopes to improve the way in which synthetic biology meets the expectations and needs of society. <br />
<br />
===Inspiration===<br />
<br />
For our Human Practice project we got inspired by a 2-year European Project called SYNBIOSAFE (1). <br />
SYNBIOSAFE was the first project in Europe to research the safety and ethical aspects of synthetic biology, aiming to proactively stimulate a debate on the following issues: safety, security, ethics and the boundary between science and society (2,3).<br />
<br />
Based on this project, we developed a complete new approach to human practice: The EPAC pyramid. Our model consists out of four elements: '''E'''ducation, '''P'''erception, '''A'''wareness & '''C'''ollaboration. These fundamentals formed the basis for organizing our activities. Because these elements are strongly related to each other we linked them together: the pyramid was born. <br />
<br />
[[Image:TU Delft Flowchart for EPAC.jpg|400px|center|thumb|Figure 1. Depending on the general public opinion and awareness, scientist can provide the right information to inform about science & technology. People recieve this information and process this according to their own values and standards. After processing this information, people will be better able to join the discussion on science & technology.]]<br />
<br />
===EPAC - This is how it works===<br />
<br />
Using our unique and holistic EPAC approach, we could really focus on the different disciplinaries involving human practice and help society to consider, guide and address the impacts of ongoing advances in synthetic biology.<br />
Working with the model makes it important to first distinguish two information streams. Information can be received and distributed (figure 1).<br />
<br />
In our point of view, we are at the interface between science and society. We can receive from and spread information to both sides. <br />
By providing information we aim to make people '''aware''' or more '''educated''' in the field of synthetic biology.<br />
The direct consequence of processing this information leads to the formation of '''perception'''. These opinions are not only based on the newly absorbed information, but also on norms, values, upbringing and education of the recipient. <br />
<br />
Therefore it is imposible to look at education, perception and awareness as separate element. There is a continuous exchange between these streams of communication.<br />
We used the principle of the adressing two of this three elements in all of our activities and thereby maintaining the connection between the elements.<br />
<br />
The C in EPAC comes from [[Team:TU_Delft/Collaboration|collaboration]]. Without cooperation, all of the above is not possible.<br />
<br />
<br />
''With the EPAC pyramid we provided a standard model for everyone that aims to manage their activities in the ongoing debate in the field of synthetic biology.''<br />
<br />
===Our activities in the EPAC===<br />
<br />
Click on the icons on the EPAC-pyramid to check our human practice activities.<br />
<br />
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<br />
===References===<br />
#'''Ganguli-Mitra, A., M. Schmidt, et al.''' (2009). Of Newtons and heretics. ''Nat Biotechnol'' 27(4): 321-322.<br />
#'''Schmidt, M., A. Ganguli-Mitra, et al.''' (2009). A priority paper for the societal and ethical aspects of synthetic biology. ''Syst Synth Biol'' 3(1-4): 3-7.<br />
#'''Schmidt, M., H. Torgersen, et al.''' (2008). SYNBIOSAFE e-conference: online community discussion on the societal aspects of synthetic biology. ''Syst Synth Biol'' 2(1-2): 7-17.</div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/sensing/resultsTeam:TU Delft/Project/sensing/results2010-10-27T20:16:05Z<p>Ptmvanboheemen: </p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing/parts" target="" /></map></html><br />
<br />
==Sensing Results==<br />
<br />
===pCaiF strength===<br />
====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF]====<br />
[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] is by far the smallest part of our project. This part enable the expression of proteins at low glucose concentrations in order to mimic a diauxic shift for the alkane degradation system: Once the preferred substrate, glucose becomes limiting, the expression of alkane degradation genes under pCaiF control will enable the cells to shift from glucose metabolism to alkane degradation. The whole regulation is performed by a piece of just 51 bp. <br />
<br />
The pCaiF regulation mechanism is really simple, [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] contains a cAMP-crp complex binding domain, cAMP-crp is known as transcriptional regulator. When the glucose concentration in the environment is high, cAMP levels are low because there is a lot of energy source that can be metabolized by the cells. However during starvation periods cAMP levels increase and thus the concentration of the complex cAMP-crp that is known to activate at least 180 genes related to starvation response. Among those pCaiF a protein used during Carnitine anaerobic metabolism. <br />
<br />
<br />
<br />
As an organism expressing our proteins using [http://partsregistry.org/Part:BBa_K398326 BBa_K398326 (pCaiF)] could not be constructed. Nevertheless, we studies the regulatory part using a proxy system with GFP. We attached a GFP generator in order to measure the output of our part. <br />
<br />
=====LB medium=====<br />
<br />
First we used a rich medium (LB) and we diluted it with M9 medium without glucose (50% v/v) in order to show the differences in protein expression when the carbon source concentration is lower in a rich medium. <br />
<br />
[[Image:TU_Delft_pCaiF_LB.jpg|600px|thumb|center| GFP and Biomass profiles in LB growth. (A) Normal LB and (B) LB diluted 50% with M9 medium without C-source.]]<br />
<br />
Our findings suggest that there is GFP production when the LB medium is diluted 50%. However the fluorescence produced is a really low signal and we didn't see an increase of GFP production during the stationary phase (starvation). This could be due to the fact that LB medium can keep the cAMP levels low for long periods of time. <br />
<br />
=====M9 minimal medium=====<br />
<br />
We then tested the response of pCaiF using minimal M9 medium at glucose concentrations of 10 g/L, 5 g/L and 2 g/L. From this experiment we expected to see a limitation of carbon source leading to a pCaiF response. <br />
[[Image:TU_Delft_pCaiF_glucose.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
In this experiment we clearly saw a significant increase in GFP production when the glucose initial concentration was 2 g/L compared to our result for an initial glucose concentration of 10 g/L; whereas at 5 g/L we found a slight increase in GFP production at the end of our measurements. From these plots we can conclude that our part is sensitive to cAMP levels as we expected. Moreover, we found that our part is active under limitation (entry of stationary phase at low glucose concentrations, see figure). <br />
<br />
[[Image:TU_Delft_pCaiF_comp.jpg|600px|thumb|center| GFP profiles in M9 medium at different initial glucose concentrations.]]<br />
<br />
=====Diauxic shift=====<br />
In order to see the effect of a second carbon source in the medium, we decided to decrease the initial glucose amount to 1 g/L and added Potassium Laurate at a final concentration of 5 mM. This experiment is very close to the later application as lauric acid is metabolized via beta oxidation. We expect to observe a diauxic shift with high expression level during the lag phase of the metabolic switch.<br />
<br />
[[Image:TU_Delft_pCaiF_diauxic.jpg|600px|thumb|center| GFP and Biomass profiles in M9 medium with glucose concentration of 1 g/L and Potassium Laurate (5mM) as secondary carbon source.]]<br />
<br />
Our findings in this experiment were really interesting, (1) the GFP signal first increases, indicating a limitation due to glucose depletion and (2) the GFP production decreases again when the catabolism of the second carbon source (lauric acid) starts. There is a clear change in the slope for the GFP profile over time with a max at about 8h.<br />
<br />
===== Does [http://partsregistry.org/Part:BBa_K398326 pCaiF] really works under starvation conditions???=====<br />
We had some doubts about the activity of pCaiF in the stationary phase because we usually observed a leaky production of GFP that followed the biomass profile. Therefore we decided to compare the GFP production during the exponential and stationary phase.<br />
<br />
[[Image:TU_Delft_pCaiF_final2.jpg|600px|thumb|center|Comparison of the GFP production rates during exponential and stationary phase.]]<br />
<br />
According to our results, there is indeed a difference between both conditions. Which is specially clear when the glucose initial concentration is 2 g/L. For the Laurate enriched medium (diauxic shift) we observed a remaining activity from the glucose limitation period and then a clear reduction during Laurate consumption.<br />
<br />
=====[http://partsregistry.org/Part:BBa_K398326 BBa_K398326 pCaiF] IN NUMBERS=====<br />
In order to convert Fluorescence arbitrary units to something meaningful, we used the parameters suggest in the part registry for [http://partsregistry.org/Part:BBa_E0040 E0040]. We divided the fluorescence random units by 79.429 (conversion factor to nM), then we calculated back the number of moles per well knowing that our reaction volume was 100 uL. If we multiply the total amount of moles per well by the [http://en.wikipedia.org/wiki/Avogadro_constant Avogadro's constant], we will get the total amount of GFP molecules produced in each data point. Then we applied a linear regression to each growth phase and we divided by the O.D. at 600nm in order to normalize the result ([http://partsregistry.org/RiPS RiPS]/O.D.). <br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th>Condition</th><th>Exponential phase [GFP molecules/O.D.] </th><th>Stationary phase [GFP molecules/O.D.]</th></tr> <tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr> <tr><td>LB</td><td>1.2516E+07</td><td>-8.6245E+05</td></tr> <tr><td>0.5LB</td><td>1.8101E+07</td><td>-2.5945E+05</td></tr> <tr><td> [glc]=10g/L</td><td>6.6456E+06</td><td>-6.7109E+06</td></tr> <tr><td>[glc]=5g/L</td><td>8.3673E+06</td><td>4.2339E+06</td></tr> <tr><td>[glc]=2g/L</td><td>7.2869E+06</td><td>3.9755E+07</td></tr> <tr><td>Diauxic growth (GLUCOSE PHASE)</TD><TD>7.4517E+06</TD><TD>2.1543E+07</TD></TR> <TR><TD>DIAUXIC GROWTH (LAURATE PHASE)</TD><TD>1.1435E+07</TD><TD>6.6428E+06</TD></TR></TABLE><br />
<br />
NOTE: CHECK AT THE PART REGISTRY THE DEFINITION OF [HTTP://PARTSREGISTRY.ORG/RIPS RIPS]<br />
<br />
=====CONCLUSIONS=====<br />
<br />
THE SIMPLE PCAIF PROMOTER WITH CRP-CAMP BINDING SITE HAS SHOWN ACTIVITY UNDER LIMITING NUTRIENT CONDITIONS. THEREFORE THIS PROMOTOR CAN BE USED TO ENABLE A CATABOLIC SHIFT FROM GLUCOSE TO NEW DEGRADATION PATHWAYS. <br />
<br />
FOR US THIS IS A VERY USEFUL PART FOR FUTURE TEAMS. <br />
PLEASE ALSO SEE THE RESULTS OBTAINED BY MODELING: <br />
[HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=MODELING/PCAIF-MODEL IN SILICO WORK]<br />
<br />
=====WORK FOR NEXT TEAM=====<br />
<br />
WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
<br />
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<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML><br />
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=====WORK FOR NEXT TEAM=====<br />
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WE WOULD HAVE LIKED TO EXPRESS OUR RESULTS ACCORDING TO THE PROTOCOLS IN [HTTP://PARTSREGISTRY.ORG/POPS POLYMERASE PER SECOND]. THEREFORE THE OUTPUT OF B0032 AND A STANDARD PROMOTER GROWING IN M9 MEDIUM HAS TO BE MEASURED.<br />
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<HTML><CENTER><IMG SRC="HTTP://2010.IGEM.ORG/WIKI/IMAGES/0/00/TU_DELFT_PROJECT_NAVIGATION.JPG" USEMAP="#PROJECTNAVIGATION" BORDER="0" /></CENTER><MAP ID="PROJECTNAVIGATION" NAME="PROJECTNAVIGATION"><AREA SHAPE="RECT" ALT="CHARACTERIZATION" TITLE="" COORDS="309,3,591,45" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/CHARACTERIZATION" TARGET="" /><AREA SHAPE="RECT" ALT="RESULTS" TITLE="" COORDS="609,3,891,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/RESULTS" TARGET="" /><AREA SHAPE="RECT" ALT="PARTS" TITLE="" COORDS="9,3,290,44" HREF="HTTP://2010.IGEM.ORG/TEAM:TU_DELFT#PAGE=PROJECT/SENSING/PARTS" TARGET="" /></MAP></HTML></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDHTeam:TU Delft/Project/alkane-degradation/results/ALDH2010-10-27T19:56:56Z<p>Ptmvanboheemen: </p>
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<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
===Characterization of the ALdehyde DeHydrogenase system===<br />
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=====Assay based on growth (Aldehydes as sole C-source)=====<br />
The recombinant strains ''Escherichia coli'' 029A ([http://partsregistry.org/Part:BBa_K398029 BBa_K398029] on the plasmid pSB1A2) and ''Escherichia coli'' 030A ([http://partsregistry.org/Part:BBa_K398030 BBa_K398030] on the plasmid pSB1A2) were culture on M9 using Octanal and Dodecanal as sole carbon sources. They didn't show visible growth after 48 hours. No further experiments using this protocol were performed afterwards.<br />
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=====Resting cell assays=====<br />
*We grew 'Escherichia coli'' 029A, ''Escherichia coli'' 030Aand ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
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*After an overnight incubation at 37ºC with the substrate (octanal), the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
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[[Image:TUDelftALDH_chrom.jpg|750px|thumb|center| Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 029A (our recombinant strain) and (D) E. coli 029A (our recombinant strain).]]<br />
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*According to our results there was a peak reduction for octanal, however we were worried because of the fact that we didn't see the peak of the product (dodecanoic acid) appearing. It could be that the cells are just storing the product inside the cell. The experiment required to much time in order to get more results: the extractions, preparation of triplicates, cultures, cell suspensions... etc. it was a work that would require an effort of all the team for a couple of weeks. We decided to go for something easier and faster to measure; since we had some experience with the NADH determinations that was our choice.<br />
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==== Enzyme activity assay ====<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was between 0.5-0.8. Two different cultures of each recominant strain and the negative control were prepared.<br />
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Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. A standard curve for protein quantification was prepared using Bradford.<br />
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The total protein of each sample was quantified using 20uL of cell extract.<br />
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The aldehyde dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanal as substrate.<br />
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After the data treatment, the results that we obtained were the following:<br />
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<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>&nbsp;</th><th>J13002</th><th>029A (1)</th><th>029A (2)</th><th>030A (1)</th><th>030A (2)</th><th>P. putida</th></tr> <tr><td>Specific activity</td><td>kat/mg</td><td>8.604E-11</td><td>1.470E-10</td><td>2.853E-10</td><td>2.433E-10</td><td>2.912E-10</td><td>6.362E-10</td></tr> <tr><td>&nbsp;</td><td>U/mg</td><td>5.162E-03</td><td>8.820E-03</td><td>1.712E-02</td><td>1.460E-02</td><td>1.747E-02</td><td>3.817E-02</td></tr> <tr><td>Standard deviation</td><td>&nbsp;</td><td>1.778E-12</td><td>6.187E-12</td><td>4.026E-11</td><td>3.539E-11</td><td>4.319E-11</td><td>3.361E-11</td></tr> <tr><td>Relative to (J13002)</td><td>&nbsp;</td><td>100.00%</td><td>170.86%</td><td>331.59%</td><td>282.79%</td><td>338.46%</td><td>739.41%</td></tr> <tr><td>Relative activity (P. putida)</td><td>&nbsp;</td><td>13.52%</td><td>23.11%</td><td>44.84%</td><td>38.25%</td><td>45.78%</td><td>100.00%</td></tr> <tr><td>T-test vs J13002</td><td>&nbsp;</td><td>-</td><td>0.9827</td><td>0.9599</td><td>0.9764</td><td>0.9976</td><td>0.9976</td></tr> <tr><td>T-test vs P. putida</td><td>&nbsp;</td><td>0.9999</td><td>1.0000</td><td>0.9998</td><td>0.9974</td><td>1.0000</td><td>-</td></tr> <tr><td>Average activity [kat/mg]</td><td>&nbsp;</td><td>8.604E-11</td><td>2.161E-10</td><td>&nbsp;</td><td>2.673E-10</td><td>&nbsp;</td><td>6.362E-10</td></tr></table><br />
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You can download a file with our raw data, results anda summary here: [[ Image:TUDelft_ALDH_results.xls]]<br />
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==== Protein expression====<br />
According to our protein gel, ''E. coli'' 030A overporduces ALDH. Whereas the protein production in ''E. coli'' 029A is barely visible. From this result we can conclude that overproduction is not necessary in order to obtain biological activity. From an overproduction of the enzyme the improvement is just 9% of the ''Pseudomonas putida'' activity. This could mean that there is protein biologically inactive, which is just a waste of cellular resources.<br />
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[[Image:TUDelftADH_gel.jpg|600px|thumb|center|Expression profile for different E. coli strains. Expected size for ALDH 53.81 kDa]]<br />
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==== Conclusion====<br />
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Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks, we couldn't prove ''in vivo'' activity. However, it is clear from the statistical analysis performed that the expression of the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. <br />
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In our resting cell experiment, apparently there is a decrease of the octanal peak, which may be due to the biological activity of our construct. However, it was not possible to see the formation of the expected product: octanoic acid. Other experiments like more resting cell assays (analyzing intracellular metabolites) or the use of C13 marked octanal may be useful in order to confirm and measure the ''in vivo'' biological activity of these parts.<br />
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[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
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*From the work published by Kato and co-workers in 2010 [http://www.springerlink.com/content/214116w7482469g0/fulltext.pdf], we knew that the purified enzyme has an activity equal to 0.63 U/mg, and according to the same study the tetradecanal dehydrogenase activity is 73 times higher than the octanal dehydrogenase; from the figure 5a of the cited paper we inferred that the dodecanal dehydrogenase activity is around 50% of the tetradecanal activity. Thus, the dodecanal dehydrogenase should be around 36.5 times the activity of octanal dehydrogenase which gives 22.995 U/mg pure ALDH. <br />
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*The same number expressed in kat/mg equals to 4e-7 kat/mg pure ALDH. Our cell extracts have dodecanal dehydrogenase activities of 2.15e-10 kat/mg protein cell extract (029A) and 2.67 e-10 kat/mg protein cell extract (030A). If we substract the normal ''E. coli'' activity, the net activities given by our biobricks are 1.301e-10 kat/mg protein cell extract (029A) and 1.812e-10kat/mg protein cell extract (030A). Which means that our strains had produced 3.253e-4 mg pure ALDH/mg protein cell extract (029A) and 4.53e-4 mg pure ALDH/mg protein cell extract (030A), respectively. Assuming a 70%(w/w) of protein content in ''E. coli'', that means that a strain carrying pSB1A2 with the biobricks [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] and [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] will produce 0.0317% (w/w) and 0.0228% (w/w) of their total weight as ALDH protein. <br />
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*From the expression profile, we saw that the strain ''E. coli'' 029A produces less ALDH protein; however, the activity is comparable to the strain ''E. coli'' 030A which over produces ALDH, this means that the strain 029A spends more efficiently its cell resources and produces a highly active ALDH protein.<br />
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==== About our project and RBS characterization (link between projects)====<br />
*Originally, we decided to characterize the Anderson promoter family because we wanted to functionally express our proteins without stressing our cells. So far, with the biobrick [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] we achieved an ''in vitro'' activity close to 34% of the activity reported for a natural alkane-degrading bateria. If someone else proves that our biobrick has the expected biological activity then that would mean that our design was successful. <br />
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*Nowadays, WE, as Synthetic Biologists, have the challenge of starting to produce parts that are being expressed in cells in the exact amount in order to confer biological activity. Over-expression (the standard protein expression method so far) is equal to waste; cells invest most of their resources for protein production (stressing the cells [http://www.springerlink.com/content/69k0pcbyc4l2qmfx/]); moreover, the production of inclusion bodies (aggregates of inactive protein) is reported when there is a high over-expression level [http://mbel.kaist.ac.kr/lab/research/protein_en1.html]. This means that high levels of protein over-expression will give as output large quantities of protein without biological function. <br />
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*With this project, we wanted to give other teams an insight about how much of specific proteins should be expressed in a cell in order to confer biological activity. Questions that arose at the beginning of our project were: How much of a protein should be expressed in order to have biological activity? Which promoter-rbs combo should be used in order to achieve a successful expression of a biologically active protein?. During the design phase (first two months), we didn't find answers to these questions and we decided to take the initiative and give the first steps towards finding answers to our questions. <br />
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*Some people may think that we didn't succeed on proving that our biobricks confer biological activity, however we feel that with this study we are giving the first steps on the way of engineering pathways using standard parts and rational expression of proteins. Some numbers about biologically active protein were given, in order to confirm our statements more studies are required and we suggest to use [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] as a starting point, ADH ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018]) is also a nice starting point because we confirmed that has biological activity; however its performance is really mediocre compared to a natural oil-degrading bacteria as ''Pseudomonas putida''.<br />
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==== What would we measure if we had more time for this project? ====<br />
'''''In vivo'' activity tests:''' A comparison of ''in vitro'' activities maybe it is not interesting from the functional point of view, since those differences could be related to substrate affinity or discrepancies in the optimal pH. C13-labeled Dodecanal degradation measurements or resting cell assays or other kinds of studies are required in order to prove the ''in vivo'' activity of our proteins.<br />
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'''Fraction of Bt-ALDH expressed compared to the total amount of protein in the cell.''' We inferred the amount of ACTIVE Bt-ALDH in our cell extract, however this number is not accurate until we can measure the total amount of Bt-ALDH in the recombinant strain.<br />
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'''Expression of Bt-ALDH using other promoter-rbs combo.''' This will give us an insight about the optimum promoter-rbs combo required for the highest possible activity without causing cell stress.<br />
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If someone else reports that our numbers and statements are correct, then they will confirm by the very first time in the iGEM competition the amount of protein required for successful expression of an ''in vivo'' biologically active enzyme. We will look forward for anyone willing to do this job on 2011 ;-)<br />
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<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADHTeam:TU Delft/Project/alkane-degradation/results/ADH2010-10-27T19:56:12Z<p>Ptmvanboheemen: </p>
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<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
===Characterization of the Alcohol DeHydrogenase (ADH) system===<br />
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=====Assay based on growth (Alcohols as sole C-source)=====<br />
*Cultures of our recombinant strain ''Escherichia coli'' 018A ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018] on the plasmid pSB1A2) showed no growth, when Octanol-1 and Dodecanol-1 (1% v/v) were used as sole C-source. No further experiments were done using this protocol.<br />
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=====Resting cell assays=====<br />
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*Cells in exponential growth phase were obtained from 50mL cultures. The cells were harevested when the O.D. at 600nm was around 0.3. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
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*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We performed the GC analysis, the chromatograms are shown below:<br />
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[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|Chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]<br />
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*No octanol-1 degradation was found ='( , after this experiment the protocol was abandoned. Probably, the long-chain alcohols cannot pass through the cell membrane and we therefore performed assays with cell extracts in order to know if there was any biological activity in the cytoplasma.<br />
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=====NADH production in cell extracts=====<br />
*'''EXPERIMENT 1'''<br />
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Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.<br />
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Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
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The total protein of each sample was quantified using 20uL of cell extract.<br />
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The alcohol dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [https://static.igem.org/mediawiki/2010/3/3c/TUDelft_ADH_raw.xls TUDelft_ADH_raw.xls]<br />
After the data treatment, the results that we obtained are shown in the table below.<br />
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[[Image:TUDelftADH_pH95.jpg|400px|thumb|center|ADH activity at pH=9.5]]<br />
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[[Image:TUDelftADH_pH8.jpg|400px|thumb|center|ADH activity at pH=8.0]]<br />
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*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. You can download our results file here: [https://static.igem.org/mediawiki/2010/8/89/TUDelft_ADH_results.xls TUDelft_ADH_results.xls]<br />
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* If you are not familiar with the term '''''katal''''', [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] for a further explanation.<br />
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*T-tests were performed in order to check the statistical significance of the difference between the wild type activity of ''E. coli'' as ADH and our recombinant strain. We set our level of significance at 0.025, in two tailed test. That means that if our t-test result is 0.95 we conclude that both samples are statistically different, whereas 0.949 or lower means that both samples are not statistically different.<br />
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<html><br />
<table><br />
<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr> <br />
<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr> <br />
<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr> <br />
<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr> <br />
<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr> <br />
<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr> <br />
<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr> <br />
<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr><br />
</table><br />
</html><br />
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*'''EXPERIMENT 2'''<br />
We ran a second experiment at pH 9.5, with two other strains that seemed to be positive in the colony PCR test. In this test we also added two extra strains of E. coli in order to have more data for the statistical analysis, the strains used were J13002 and 331 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331] in pSB1A2). We followed the same procedure as in the experiment 1, for this experiment we also included a positive control which was cell extracts from a culture of ''Pseudomonas putida'' growing on octane as sole carbon source, the cells were inoculated the night before and they were harvested when the O.D. at 600 nm was 0.608; the results obtained were the following:<br />
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<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>kat/mg</th><th>U/mg</th><th>STDEV</th><th>Improvement</th><th>T-test</th><th>Relative to putida</th><th>&nbsp;</th></tr> <tr><td>E. Coli</td><td>8.75E-12</td><td>5.25E-04</td><td>8.18987E-12</td><td>-</td><td>-</td><td>0.90%</td><td>&nbsp;</td></tr> <tr><td>018A-2</td><td>9.30E-12</td><td>5.58E-04</td><td>1.84877E-12</td><td>6.26%</td><td>0.4708</td><td>0.96%</td><td>&nbsp;</td></tr> <tr><td>018A-3</td><td>3.05E-11</td><td>1.83E-03</td><td>6.27802E-12</td><td>249.14%</td><td>0.9808</td><td>3.15%</td><td>&nbsp;</td></tr> <tr><td>018A-4</td><td>8.26E-12</td><td>4.96E-04</td><td>1.8256E-12</td><td>-5.59%</td><td>0.4053</td><td>0.85%</td><td>&nbsp;</td></tr> <tr><td>P. Putida</td><td>9.69E-10</td><td>5.82E-02</td><td>2.09109E-10</td><td>-</td><td>0.9883</td><td>100.00%</td><td></td></tr></table><br />
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According to these results, the other two strains (018A-2 and 018A-4) do not functionally express the part BBa_K398018. However, these results confirm our findings from the first experiment. You can download our result in this link: [[ Image:TUDelft_ADH_results2.xls]] You can also find a summary in this file:[[ Image:TUDelft_ADH_summary.xls]]<br />
<br />
==== Conclusions ====<br />
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[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
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According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to our analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95, which means that the part [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the parts [http://partsregistry.org/Part:BBa_K398005 BBa_K398005] and [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] have catalytic activity; particularly when we used [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] the enzyme activity of ''E. coli'' cell extracts was equivalent to 3% of the ''in vitro'' activity of the positive control (''Pseudomonas putida''). <br />
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However, we think that from this comparison of both ''in vitro'' activities we can suggest future teams interested in our part [http://partsregistry.org/Part:BBa_K398005 BBa_K398005] to use a stronger promoter-rbs combination; maybe by increasing the amount of protein produced it is possible to get higher ''in vitro'' and ''in vivo'' activities. Nevertheless, we have to stress that the ''in vivo'' activity of the part [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] is still unknown for us, maybe there is a lack of long-chain alcohol transporter proteins or it is necessary to over-express this part in order to see/measure it.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadATeam:TU Delft/Project/alkane-degradation/results/LadA2010-10-27T19:55:47Z<p>Ptmvanboheemen: </p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
===Characterization of the long-chain alkane monooxygenase; LadA===<br />
====Enzyme activity assay based on NADH absorbance====<br />
Curves of the NADH absorbance for the blank, negative control and strains of interest displayed no significant differences, thus this method was abandoned. The reason for this could be explained by a lack of cofactors, a substrate diffusion limitation or even substrate inhibition.<br />
<br />
====Enzyme activity assay based on GC-analysis====<br />
Our enzyme activity [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Enzyme_activity_assay_for_LadA_by_GC protocol] for LadA was loosely based an article by Maeng et al,1996. Homogenization of the alkane in the 50mM Tris buffer was achieved by adding Triton-X100, boiling and sonication. After 12 hours the residual alkane was extracted using EtOAc.<br />
The following are typical examples of the chromatographs obtained:<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:TUDelft_Blank C-16.png|400px|thumb|left|Chromatograph of blank containing hexadecane]]<br />
<br />
[[Image:TUDelft_J13.png|400px|left|thumb|Chromatograph of the negative control for the enzyme kinetics of the alkane monooxygenase. The negative control consisted of ''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] in pSB1A2.]]<br />
<br />
[[Image:TUDelft_017-7.png|400px|left|thumb|Chromatograph of sample obtained from ''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398017 BBa_K398017] in pSB1A2.]]<br />
<br />
[[Image:TUDelft_027.png|400px|left|thumb|Chromatograph of sample obtained from ''E.coli'' TOP10 carrying [http://partsregistry.org/Part:BBa_K398027 BBa_K398027] in pSB1A2.]]<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|-<br />
|~20.0<br />
|Hexadecane (substrate)<br />
|-<br />
|}<br />
<br />
The analysis of the GC graphs was analogous to that of the AH system, described above. We made use of the internal standards present in each sample to determine the ratio of the surface areas of hexadecane vs. internal standard. These ratios can be related to the amounts of hexadecane added to eventually estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
====Conclusions====<br />
LadA was characterized succesfully using an enzyme kinetics assay coupled to gas chromatography. We see a significant increase in enzyme activity in the strains carrying the ladA protein generator BioBrick compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein compared to 0.55E-03 of the negative control strain. <br />
Further studies into the enzymatic activity needed to sustain growth could be performed in future. This will give us information on the implementation of this BioBrick into our complete alkane degradation chassis.</div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylaseTeam:TU Delft/Project/alkane-degradation/results/alkane hydroxylase2010-10-27T19:55:05Z<p>Ptmvanboheemen: </p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
===Characterization of the alkane hydroxylase system===<br />
====Growth analysis====<br />
We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.<br />
<br />
====Resting-cell assays====<br />
The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].<br />
<br />
The following are examples of typical chromatographs obtained:<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:Blank_C-8.png|400px|thumb|left|Chromatograph of blank containing octane.]]<br />
<br />
[[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]<br />
<br />
[[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~5.2<br />
|Octane (substrate)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|}<br />
<br />
The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. Using a two-point calibration method, where we set the ratio of 0.898 equal to the total amount of octane added (16.2 uL, 99.7 umol), we can estimate the enzymatic activity of the AH-system. We know the reaction time (12 h.) and the mg of protein for each sample (using the conversion factor 0.41g/L/OD). See the table below for details.<br />
<br />
====Conclusions====<br />
We've succeeded in characterizing the activity of the alkane hydroxylase system using gas chromatography. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system. <br />
The lack of an octanol peak in the GC-graphs above could be caused by the octanol being trapped by the cell, and thus not showing up in the organic (EtOAc) phase. Another likely explanation is that the octanol is readily converted by an aspecific alcohol dehydrogenase native to the ''E.coli'' K12 strain.<br />
In the future we would like to perform a resting-cell assay using ''P. putida'' and determine the enzymatic activity needed to support growth. A comparison of this natural-degrader value with the biological activity we've found of 0.045 U/mg would give more information on the growth feasibility of a strain carring the AH-system BioBrick on octane.<br />
Furthermore, a study could be done on the AH-system over time to find out more information on the kinetics of the system.</div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradationTeam:TU Delft/Project/alkane-degradation2010-10-27T19:54:30Z<p>Ptmvanboheemen: </p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Alkane Degradation==<br />
[[Image:TUDelft_Degradation.png|300px|right]]<br />
Pollution of soil and water environments by crude oil has been, and is still today, an important environmental issue. Crude oil is a complex mixture of thousands of compounds, of which alkanes constitute the major fraction. Alkanes are saturated hydrocarbons of different sizes and structures. Although they are chemically relatively inert, several micro-organisms can efficiently degrade most of them. Literature research has revealed a number of genes that are most likely responsible for this degradation. These genes will be implemented into ''E.coli'' using the BioBrick standard and their alkane degrading capabilities will be characterized. On the alkane degradation a [https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA metabolic flux analysis] was performed to determine the maximal yield for biomass growth on alkanes.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===Continue reading===<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:53:59Z<p>Ptmvanboheemen: /* USEFUL LITERATURE AND REFERENCES */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. <br />
<br />
If you want to know more about of our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ALDH|detailed ALDH results]] page. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====Useful Literature and References====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:53:29Z<p>Ptmvanboheemen: /* Characterization of the ALdehyde DeHydrogenase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. <br />
<br />
If you want to know more about of our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ALDH|detailed ALDH results]] page. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:52:32Z<p>Ptmvanboheemen: /* Characterization of the Alcohol DeHydrogenase (ADH) system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/ADH|detailed ADH results]] page.<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:51:48Z<p>Ptmvanboheemen: /* Characterization of the alkane hydroxylase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:51:33Z<p>Ptmvanboheemen: /* Characterization of the alkane hydroxylase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [[Project/alkane-degradation/results/alkane_hydroxylase|detailed alkane hydroxylase results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:51:04Z<p>Ptmvanboheemen: /* Characterization of the long-chain alkane monooxygenase; LadA */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase detailed alkane hydroxylase results] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, read the [[Team:TU_Delft/Project/alkane-degradation/results/LadA|detailed LadA results]] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/resultsTeam:TU Delft/Project/alkane-degradation/results2010-10-27T19:50:06Z<p>Ptmvanboheemen: /* Characterization of the alkane hydroxylase system */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase Characterization of the alkane hydroxylase system]===<br />
<br />
The following table contains the average ratio of the hexadecane/undecane surface areas. Strains numbers 1 and 2 are two positive colonies taken from the same plate. <br />
From the ratios we may conclude that the samples obtained from the ''E.coli'' strain, carrying the AH system, contain relatively less octane than the control strain. By comparing peak ratios we were able to estimate the specific enzymatic activity of the system, which was found to be 0.045 U/mg. <br />
<br />
For more information about our findings, read the [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/alkane_hydroxylase detailed alkane hydroxylase results] page.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Ratio octane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Octane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.832<br />
|6.16<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control<br />
|0.821<br />
|13.3<br />
|1.34<br />
|1.51<br />
|1.23E-3<br />
|-<br />
|''E.coli'' K12 strain #1 (AH-system)<br />
|0.652<br />
|14.4<br />
|21.6<br />
|1.70<br />
|1.77E-2<br />
|-<br />
|''E.coli'' K12 strain #2 (AH-system)<br />
|0.439<br />
|3.96<br />
|47.0<br />
|1.46<br />
|4.49E-2<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA Characterization of the long-chain alkane monooxygenase; LadA]===<br />
<br />
The analysis of the GC graphs allowed us to estimate the enzymatic activity. The following table displays the average ratios found and the enzyme activity estimates. Strains numbers 1 and 2 are two positive colonies taken from the same plate.<br />
<br />
<br />
We observed a significant increase in enzyme activity in the strains carrying the ladA protein generator compared to the negative control strain. The highest enzyme activity value was found to be 3.33E-03 U/mg protein. In order to know more about the characterization of this system, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/LadA CLICK ON THIS LINK]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Average ratio hexadecane/undecane</b><br />
|<b>Standard deviation [%]</b><br />
|<b>Hexadecane converted [umol]</b><br />
|<b>Protein [mg]</b><br />
|<b>Enzymatic activity [U/mg protein]<br />
|-<br />
|Blank<br />
|0.900<br />
|3.19<br />
|0.00<br />
|0.00<br />
|0.00<br />
|-<br />
|''E.coli'' K12 negative control ([http://partsregistry.org/Part:BBa_J13002 J13002])<br />
|0.807<br />
|3.48<br />
|1.76<br />
|4.46<br />
|5.49E-04<br />
|-<br />
|''E.coli'' K12 strain #1 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.600<br />
|15.4<br />
|5.68<br />
|4.58<br />
|1.72E-03<br />
|-<br />
|''E.coli'' K12 strain #2 ([http://partsregistry.org/Part:BBa_K398017 K398017])<br />
|0.532<br />
|4.25<br />
|6.95<br />
|3.18<br />
|3.03E-03<br />
|-<br />
|''E.coli'' TOP10 strain ([http://partsregistry.org/Part:BBa_K398027 K398027])<br />
|0.492<br />
|2.98<br />
|7.71<br />
|3.22<br />
|3.33E-03<br />
|}<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH Characterization of the Alcohol DeHydrogenase (ADH) system]===<br />
According to our results, the ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain expressing the Biobrick [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] has an activity of 2.93e-11 kat/mg (1.76 mU/mg), an improvement of 2-fold compared to the wild type activity; which also means 3% of the activity of the positive control ''Pseudomonas putida''.<br />
<br />
If you are interested in knowing more about our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ADH CLICK ON THIS LINK].<br />
<br />
[[Image:TUDelftADH_final.jpg|600px|thumb|center|Comparison between E. coli ADH activity and our recombinant strain. ]]<br />
<br />
===[https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH Characterization of the ALdehyde DeHydrogenase system]===<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks. The expression of ALDH under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold; whereas the expression of the same protein using the part [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] as promoter-rbs combo increases the same activity 3-fold. <br />
<br />
Moreover, the enzymatic activities measured for the constructs [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] and [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] were equivalent to 33.98% and 42.01% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity, respectively. If you want to know more about of our findings, [https://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results/ALDH CLICK ON THIS LINK]. <br />
<br />
It is worthy to mention that our part expresses the protein in a lower amount, meaning that cells express a highly active protein. Thus the cellular resources are spent in a more efficient way than in the strain that overproduces ALDH.<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px|thumb|center|Comparison of ALDH activities in the different strains tested in this study]]<br />
<br />
<br />
====USEFUL LITERATURE AND REFERENCES====<br />
#Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading ''Geobacillus thermoleovorans'' B23" Extremophiles (2010) 14:33-39.<br />
#http://mbel.kaist.ac.kr/lab/research/protein_en1.html<br />
#Hoffmann F. and Rinas U. "Stress Induced by Recombinant Protein Production in ''Escherichia coli''" Advances in Biochemical Engineering/Biotechnology, 2004, Vol. 89/2004, pp. 73-92.<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>Ptmvanboheemenhttp://2010.igem.org/Team:TU_Delft/Project/solubility/resultsTeam:TU Delft/Project/solubility/results2010-10-27T19:34:44Z<p>Ptmvanboheemen: /* Conclusions */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
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<br />
==Solubility Results & Conclusions==<br />
<br />
Our goal is to enhance the solubility of alkanes in water. Therefore we constructed a new part that expresses the emulsifier protein [[Team:TU_Delft/Project/solubility/alna|AlnA]] after induction by IPTG. By using our own [[Team:TU_Delft/Project/solubility/characterization|emulsification assay]] we measured the increase of hydrophobic compounds in the water phase.<br />
<br />
===Results===<br />
For the production of large amounts of protein, 1 L shake flasks cultures were induced with IPTG after the OD measuremnts indicated the start of the exponential phase. The experiment was done performed three times with iterative improvements.<br />
<br />
====Experiment 1====<br />
In this experiment we used a large volume (1 L) at 37 C. The OD measurements at the induction time point and harvest are shown in Table 1. <br />
<br />
'''Table 1:''' ''OD600 of the cultures at IPTG induction and harvest.''<br />
{|<br />
|'''#'''<br />
|'''Culture'''<br />
|'''OD600 at induction'''<br />
|'''OD600 at harvest'''<br />
|-<br />
|1<br />
|Control (J13002) 37 C<br />
|0.367<br />
|0.882<br />
|-<br />
|2<br />
|AlnA (K398206) 37 C<br />
|0.434<br />
|0.880<br />
|}<br />
<br />
The total volume of sample at the end after completion of the isolation protocol was 3 mL. The protein concentration was determined by Bradford analysis and showed that the control contained 19 mg mL<sup>-1</sup> and the AlnA sample contained 16 mg mL<sup>-1</sup>.<br />
<br />
The emulsification capacity of the protein mixture was determined using our emulsifier assay. 0.75 mg protein was used in the assay displayed in Figure 2. The 30% increase in absorption in the AlnA sample indicates a higher amount of Sudan II that is in solution, thus a higher emulsification by the proteins. A statistical T test shows that this is a significant increase (p = 3 x 10<sup>-3</sup>).<br />
<br />
[[Image:TU_Delft_Emuls_assay_exp1.jpg|thumb|400px|center|''Figure 1'': Emulsification of Sudan II by the isolated proteins from the control and AlnA culture. The measurements are corrected for background absorption caused by the sample itself and dissolved Sudan II without the addition of protein. The assay was done in triplo.]]<br />
<br />
====Experiment 2====<br />
The second experiment was carried out at two temperatures: 37 C and 30 C. It was expected that IPTG induction is stronger at lower temperatures. The OD measurements at the induction time point and harvest are shown in Table 2. <br />
<br />
'''Table 2:''' ''OD600 of the cultures at IPTG induction and harvest.''<br />
{|<br />
|'''#'''<br />
|'''Culture'''<br />
|'''OD600 at induction'''<br />
|'''OD600 at harvest'''<br />
|-<br />
|1<br />
|Control (J13002) 30 C<br />
|0.073<br />
|0.451<br />
|-<br />
|2<br />
|Control (J13002) 37 C<br />
|0.088<br />
|0.810<br />
|-<br />
|3<br />
|AlnA (K398206) 30 C<br />
|0.060<br />
|0.393<br />
|-<br />
|4<br />
|AlnA (K398206) 37 C<br />
|0.067<br />
|0.624<br />
|}<br />
<br />
The total volume of sample at the end after completion of the isolation protocol was 1 mL. The protein concentration was determined by Bradford analysis and showed that from the culture grown at 37 C the control contained 1.7 mg mL<sup>-1</sup> and the AlnA sample contained 1.5 mg mL<sup>-1</sup>. The control samples from the culture grown at 30 C contained 2.41 mg mL<sup>-1</sup> and the AlnA sample contained 2.29 mg mL<sup>-1</sup>.<br />
<br />
The emulsification capacity of the samples was determined using our emulsifier assay. 0.75 mg protein was used in the assay displayed in Figure 2. The 13% increase in absorption in the AlnA samples indicates a higher amount of Sudan II that is in solution, thus a higher emulsification by the proteins.<br />
<br />
[[Image:TU_Delft_Emuls_assay_exp2.jpg|thumb|800px|center|''Figure 2'': Emulsification of Sudan II by the isolated proteins from the control and AlnA culture grown at 37 C and 30 C. The measurements are corrected for background absorption caused by the sample itself and dissolved Sudan II without the addition of protein.]]<br />
<br />
====Experiment 3====<br />
The experiments were repeated one last time to confirm the observed emulsification capacity. The experiment was again carried out at 37 C and 30 C. The OD measurements at the induction time point and harvest are shown in Table 3. <br />
<br />
'''Table 3:''' ''OD600 of the cultures at IPTG induction and harvest.''<br />
{|<br />
|'''#'''<br />
|'''Culture'''<br />
|'''OD600 at induction'''<br />
|'''OD600 at harvest'''<br />
|-<br />
|1<br />
|Control (J13002) 30 C<br />
|0.111<br />
|0.462<br />
|-<br />
|2<br />
|Control (J13002) 37 C<br />
|0.119<br />
|0.700<br />
|-<br />
|3<br />
|AlnA (K398206) 30 C<br />
|0.110<br />
|0.435<br />
|-<br />
|4<br />
|AlnA (K398206) 37 C<br />
|0.113<br />
|0.616<br />
|}<br />
<br />
The total volume of sample at the end after completion of the isolation protocol was 1 mL. The protein concentration was determined by Bradford analysis and showed that from the culture grown at 37 C the control contained 11.33 mg mL<sup>-1</sup> and the AlnA sample contained 8.15 mg mL<sup>-1</sup>. The control samples from the culture grown at 30 C contained 5.49 mg mL<sup>-1</sup> and the AlnA sample contained 8.49 mg mL<sup>-1</sup>.<br />
<br />
The emulsification capacity of the samples was determined using our emulsifier assay. 0.75 mg protein was used in the assay displayed in Figure 2. The 26% increase in absorption in the AlnA samples at 30 C and 30% observed for the extract from cultures grown at 37 C indicate a higher amount of Sudan II that is in solution, thus a higher emulsification by the proteins.<br />
<br />
[[Image:TU_Delft_Emuls_assay_25_10_10.jpg|thumb|800px|center|''Figure 3'': Emulsification of Sudan II by the isolated proteins from the control and AlnA culture grown at 37 C and 30 C. The measurements are corrected for background absorption caused by the sample itself and dissolved Sudan II without the addition of protein.]]<br />
<br />
===Conclusions===<br />
For enabling ''E. coli'' to degrade hydrocarbons we equipped the cells with the ability to produce, AlnA, a known emulsifying protein. The production of the protein was induced by IPTG. Although known as a strong inducer, we were not able see the protein on an SDS-PAGE gel. This is probably due to a low protein synthesis of the cells when cultured in minimal medium. <br />
<br />
Nevertheless the emulsification assays do show an increased emulsifying capacity by the expression of BBa_K398206. With our emulsifier assay we determined that an increased amount of hydrophobic Sudan II is dissolved compared to the control strain with BBa_J13002. According to our calibration curve with SDS, this increase corresponds to the emulsification capacity of 8 mM of SDS.<br />
<br />
The use of cell extracts is not a final proof for AlnA activity, we can assume that the increased emulsification capacity is caused by the production of AlnA.<br />
<br />
===Work for next year teams===<br />
<br />
* In further research we would advise to add a tag to the protein, so it can be tracked and isolated with greater ease and purity.<br />
* New research should also include the emulsification of alkanes. The lipophilicity of Sudan II (Log P of 6.00 [1]) is the equal to that of dodecane and about 1.5 times that of octane [2]. So, similar results are expected.<br />
<br />
===References===<br />
# Kallury et al (2007), Solid Phase Extraction for Detection of Sudan Dye Contaminants in Spices with HPLC/UV Detection, THE APPLICATION NOTEBOOK<br />
# Jeppsson (1975), Parabolic Relationship between Lipophilicity and Biological Activity of Aliphatic Hydrocarbons, Ethers and Ketones after Intravenous Injections of Emulsion Formulations into Mice, Acta pharmacol. et toxicol<br />
<br />
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