Team:Tokyo-NoKoGen/photocontrol

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			<td height=113 bgcolor="#0082ef"><p class="style36"><strong>Tokyo-NokoGen 2011</strong></p>
			<p class="style34 f-lp">Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology</p>
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			<td height=357 rowspan=2 bgcolor="#cdcdcd"><p class="style3">&nbsp;</p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen"><span class="style18" style="text-decoration:underline">Home</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Team"><span class="style18" style="text-decoration:underline">Team</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/memebers"><span class="style18" style="text-decoration:underline">Members</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Project"><span class="style18" style="text-decoration:underline">Project: EcoLion</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/Parts"><span class="style18" style="text-decoration:underline">BioBricks</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/notebook"><span class="style18" style="text-decoration:underline">Notebook</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="Protocols"><span class="style18" style="text-decoration:underline">Protocols</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="attribution"><span class="style18" style="text-decoration:underline">Attribution</span></a></span></strong></p>
			<p class="style3"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/safety"><span class="style18" style="text-decoration:underline">Safety</span></a></span></strong></p>
			<p class="style3 f-lp"><strong><span class="style14"><a href="https://2011.igem.org/Team:Tokyo-NoKoGen/sponsors"><span class="style18" style="text-decoration:underline">Sponsors</span></a></span></strong></p>
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						<td height=676 colspan=2><p class="style63">Photocontrol</p>
						<p class="style41"><span class="style62">- phototaxis, aggregation, and lysis</span></p>
						<p class="style43">&nbsp;</p>
						<p class="style64">Introduction</p>
						<p class="style43">After having collected heavy metals using transporters and metallothioneins, and storing them inside the BMC, comes the last part of the project &#8211; how do we collect <em>E. coli</em> that has absorbed the toxic compounds? The whole procedure will be done in a large scale, and so we should think of an efficient and a convenient way for collecting the <em>E. coli</em>. The word efficiency is often used for robots and machines. They make our life convenient, by making us do less work. It would be fun if we can make <em>E. coli</em> become convenient for us, and can make them easy to control and make them move like robots and machines. An input used for robots and machines are electricity, but what can we use for <em>E. coli</em> as an input for movements? Today, there are several research and reports on <em>E. coli </em>reacting to light, a word known as phototaxis. </p>
						<p class="style43">Phototaxis is a property of <em>E. coli,</em> that allows movement of <em>E. coli</em> reacting to light. Last year we have found a few reports that have tried to engineer <em>E. coli</em> to add phototaxis, research based on Halophilic archaea [1, 2]. We have referred to the reported papers and tried creating a phototaxis device for collecting <em>E. col</em>i in an efficient way. By using light, it is possible to control the movement of <em>E. coli</em> and we can use it for collection in our system</p>
						<p class="style43"><img src="https://static.igem.org/mediawiki/2011/4/4a/Phototaxisfig1.jpg" border=0 width=256 height=293 alt="phototaxisfig1" style="vertical-align:baseline"></p>
						<p class="style43">Aggregation, is another possible option for collecting E. coli. Inducing E. coli to produce aggregation proteins Antigen43, we can make the E. coli aggregate and collect them at once as a cluster (Fig.2).</p>
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						<td height=207><img src="https://static.igem.org/mediawiki/2011/9/91/Phototaxisfig2.jpg" border=0 width=374 height=207 alt="phototaxisfig2" style="float:left"></td>
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						<td height=4755 colspan=2><p class="style43 f-fp">What we need to put in mind as a consideration is &#8211; what do we do when we fail to collect some of the E. coli? To solve the problem, we have decided to introduce lysis genes in to the E. coli. The whole procedure for collecting heavy metals from contaminated water can be done at night in the dark. The collection of heavy metals and the E. coli should be finished by the time the sun comes out. Lysis genes will be turned on by red light, leading to the death of E. coli that has leaked out from phototaxis or aggregation (Fig.3). By introducing lysis as a consideration for biosafety, we can avoid biohazard.</p>
						<p class="style43">&nbsp;</p>
						<p class="style43"><img src="https://static.igem.org/mediawiki/2011/0/0e/Phototaxisfig3.jpg" border=0 width=416 height=271 alt="phototaxisfig3" style="vertical-align:baseline"></p>
						<p class="style59">&nbsp;</p>
						<p class="style59">&nbsp;</p>
						<p class="style61">1. Phototaxis</p>
						<p class="style55"><span class="style53">1-1. Construct</span></p>
						<p class="style59"><img src="https://static.igem.org/mediawiki/2011/4/44/Phototaxisfig4.jpg" border=0 width=435 height=119 alt="phototaxisfig4" style="vertical-align:baseline"></p>
						<p class="style59">&nbsp;</p>
						<p class="style59"><strong><span class="style53">1-2. How it works</span></strong></p>
						<p class="style59"><img src="https://static.igem.org/mediawiki/2011/e/e5/Phototaxisfig5.jpg" border=0 width=404 height=333 alt="phototaxisfig5" style="vertical-align:baseline"></p>
						<p class="style59">When light is shone, CheA phosphorylates itself, which then phosphorylates CheY. CheY then changes the movement of flagellar, and makes phototaxis happen. In the dark the E. coli swims straight, but when there is light, E. coli starts tumbling and as a result moves as if it is moving away from the light.</p>
						<p class="style59">&nbsp;</p>
						<p class="style61">1-3. Evaluation</p>
						<p class="style59">We expressed phototaxis device (BBa_K317028) in E.coli DH5&#945; and evaluated its function. The E. coli was pre-cultured in 3 mL LB medium containing Chloramphenicol at 37oC until the OD660 reached 0.5. 1 mL of the pre-culture was removed into a tube, where all-trans retinal (f.c. 2 &#956;M) was added. The tubes were wrapped in aluminium foil to avoid light, and were cultured by shaking at 37 oC for 2 hours. 10 &#956;L of pre-cultured solution was plotted onto semisolid medium of 0.5% agar and 2 &#956;M all-trans retinal, and incubated overnight under light (fluorescent light) or dark (wrapped in aluminium foil) conditions. As a result, we expect to see that the size of the colony will not change under light condition because the cells will not move, but under dark condition, we expect to see that the colonies grow larger because of the induced movement.</p>
						<p class="style59">&nbsp;</p>
						<p class="style59">&nbsp;</p>
						<p class="style61">2. Aggregation</p>
						<p class="style61">2.-1 Construct	</p>
						<p class="style55"><img src="https://static.igem.org/mediawiki/2011/a/a3/Phototaxisfig6.jpg" border=0 width=355 height=116 alt="phototaxisfig6" style="vertical-align:baseline"></p>
						<p class="style55">&nbsp;</p>
						<p class="style55"><span class="style53">2</span><span class="style45">-</span><span class="style53">2. How it works</span></p>
						<p class="style59"><img src="https://static.igem.org/mediawiki/2011/2/25/Phototaxisfig7.jpg" border=0 width=458 height=334 alt="phototaxisfig7" style="vertical-align:baseline"></p>
						<p class="style59">&nbsp;</p>
						<p class="style59"><strong><span class="style53">2-3. Evaluation</span></strong></p>
						<p class="style59">The E. coli cells containing Antigen43 gene was pre-cultured in 3 mL LB medium at 37 oC for 12 hours. After adjusting its OD595, they were pre-cultured solution were suspended into 100 mL LB medium and cultured at 37 oC. Its OD595 was measured every 1 hour until the OD595 reached 2.5, and 3 mL of it was put into a test tube for IPTG induction of Antigen43 expression. After 2 hours of culturing at 37 oC, OD595 of the liquid culture 1cm from the surface was measured and plotted against a curve.</p>
						<p class="style55">&nbsp;</p>
						<p class="style55">&nbsp;</p>
						<p class="style61">3. Lysis</p>
						<p class="style55"><span class="style53">3</span><span class="style45">-</span><span class="style53">1. Construct </span></p>
						<p class="style59"><img src="https://static.igem.org/mediawiki/2011/6/6f/Phototaxisfig8.jpg" border=0 width=369 height=187 alt="phototaxisfig8" style="vertical-align:baseline"></p>
						<p class="style59">&nbsp;</p>
						<p class="style55"><span class="style53">3</span><span class="style45">-</span><span class="style53">2. How it works</span></p>
						<p class="style59"><img src="https://static.igem.org/mediawiki/2011/e/ea/Phototaxisfig9.jpg" border=0 width=458 height=241 alt="phototaxisfig9" style="vertical-align:baseline"></p>
						<p class="style59">&nbsp;</p>
						<p class="style59"><strong><span class="style53">3-3. Evaluation</span></strong></p>
						<p class="style59">Lysis genes with and without antiholin was evaluated to see any leakage of lysis gene expression. An E. coli containing an empty vector as a control, an E. coli containing Lysis genes with antiholin, and an E. coli containing Lysis genes without antiholin were cultured at 37 oC and OD660 was measured every 30min. to 1 hour.</p>
						<p class="style59">To find out if IPTG expression of lysis gene work, using the vectors containing holin and endolysin expressed under lac promoter, and antiholin expressed under a constitutive promoter, we transformed E. coli DH5&#945;. The obtained colony pre-cultured overnight in 2 mL LB and 1 mL of it was cultured in 100 mL LB at 37&#8451;. At OD660, IPTG (f.c. 3 mM) was added to induce the expression of Lysis genes. </p>
						<p class="style55">&nbsp;</p>
						<p class="style55">&nbsp;</p>
						<p class="style61">4. Results&amp;Discussion</p>
						<p class="style61">4-1. Phototaxis</p>
						<p class="style59">&nbsp;</p>
						<p class="style59">&nbsp;</p>
						<p class="style59">&nbsp;</p>
						<p class="style59">&nbsp;</p>
						<p class="style55"><span class="style53">4-2. Aggregation</span></p>
						<p class="style55"><img src="Resources/phototaxisfig10.jpeg" border=0 width=326 height=215 alt="phototaxisfig10" style="vertical-align:baseline"></p>
						<p class="style59">This is the result of antigen43 expression (fig.11).</p>
						<p class="style59">The red and orange line represents growth curve of cells containing Antigen43 under PLlacO1, and the green and the blue lines represents growth curve of cells containing no Antigen43 gene. There is a significance difference between cells with and without the aggregation proteins, as the OD595 went down just went down to a third within an hour. The OD595 of the cells for control remained stable. However, we could not see the difference between the IPTG induced cells and non-induced cells. This data could suggest that the expression of Antigen43 is not repressed well enough, and is leaking even without being induced. It might also indicate that aggregation can even happen with a small expression.</p>
						<p class="style59">&nbsp;</p>
						<p class="style55"><span class="style53">4-3. Lysis</span></p>
						<p class="style59"><img src="Resources/phototaxisfig11.jpeg" border=0 width=401 height=230 alt="phototaxisfig11" style="vertical-align:baseline"></p>
						<p class="style59">This is the result of comparison of lysis gene expression with and without antiholin (fig.12). The yellow line represents control, vector without lysis gene. The blue line shows lysis gene with antiholin and the red line shows lysis gene without antiholin. As you can see, both blue and red line show lower OD660 than the control, indicating that there might be a leakage of lysis gene expression. However system with antiholin kept a higher OD660 than the system without antiholin. Therefore, we concluded that the inducible lysis system consisting of T4 endolysin and holin, functions better with the presence of antiholn.</p>
						<p class="style59"><img src="Resources/phototaxisfig12.jpeg" border=0 width=415 height=211 alt="phototaxisfig12" style="vertical-align:baseline"></p>
						<p class="style59">This is the result of lysis gene expression with antiholin, induced by IPTG at stationary phase (fig.13). Both of these cultivation curves indicate E. coli with lysis genes with antiholin. IPTG was not added to the yellow culture, and IPTG (f.c. 3mM) was added to the blue culture. Within 2 hours after addition of IPTG, the OD660 went down by 80%, indicating that the lysis genes were successfully induced by IPTG. </p>
						<p class="style59">&nbsp;</p>
						<p class="style59">&nbsp;</p>
						<p class="style61">5. Summary</p>
						<p class="style55">&nbsp;</p>
						<p class="style55">&nbsp;</p>
						<p class="style55">&nbsp;</p>
						<p class="style61">6. Reference</p>
						<p class="style59">[1] Jung et al. (2001) An Archeaeal photosignal-Transducing Module Mediates Phototaxis in <em>Escherichia coli. J. Bacteriol.,</em> 183, 6365-6371.</p>
						<p class="style59">[2] Vishwa et al. (2003) Photostimulation of a Sensory Rhodopsin ll/Htrll/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro. <em>Biochemistry,</em> 42, 13887-13892.</p>
						<p class="style59">[3] Kjærgaard et al. (2000) Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens. <em>J. Bacteriol</em>., 182 (17) 4789&#8211;4796.</p>
						<p class="style59">[4] Young et al. (2000) Phages will out: strategies of host cell lysis. <em>Trends Microbiol,</em> 8 (3) 120-8.</p>
						<p class="style59">[5] Tran et al. (2005) Periplasmic Domains Define Holin-Antiholin Interactions in T4 Lysis Inhibition. <em>J. Bacteriol.</em>, 187 (19) 6631-6640.</p>
						<p class="style59 f-lp">[6] Engebrechi and Silverman (1984) Identification of genes and gene products necessary for bacterial bioluminescence. <em>Proc. Natl. Acad. Sci. USA</em>., 81 (13) 4154-4158.</p>
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