http://2013.igem.org/wiki/index.php?title=Special:Contributions/Barao&feed=atom&limit=50&target=Barao&year=&month=2013.igem.org - User contributions [en]2024-03-28T23:08:31ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_WorkTeam:HokkaidoU Japan/Shuffling Kit/Future Work2013-10-29T02:36:33Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E. coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
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<h1>Maestro <span class="italic">E. coli</span> Future Work</h1><br />
<br />
<h2>To make Promoter Selector better kit</h2><br />
<p>To make Promoter Selector better, we are going to place pigment producing construct between two PstI sites. After you confirm the best promoter, you can remove the followed constructs by digesting with PstI.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/12/Fig11_new2_800_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 How to make our kit better.</span></div><br />
</div><br />
<br />
<h2>Application of RBS Selector</h2><br />
<p>We can get a lot of operons by RBS Selector. RBS Selector provides 16 operons in case of 2 CDS, 64 operons in case of 3 CDS. What can we do using these number of operons? We expect industrial applications of RBS Selector as future works.</p><br />
<br />
<p><br />
In the case of producing valuable molecules inside the cell, it is important to select the RBS which results the largest yield.<br />
<br />
<br />
For example PHB <br />
(polyhydroxybutyrate) ,3 enzymes synthesize PHB from substrate. PHB is one of the eco-friendly, biodegradable plastic. You can learn more about PHB at <a href="https://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis">iGEM HokkaidoU_Japan 2012 wiki</a>. In the production of PHB, each CDS has the optimum RBS. Our RS can be used for selecting the best combination of RBSs.<br />
<br />
<br />
<br />
It is possible to use RBS Selector when producing medicine or vaccine etc.<br />
<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2012/3/38/HokkaidoU_PHB_Fig3.jpg"><br />
<div><span class="bold">fig.2 P(3HB) synthesis pathway in R. eutropha.</span></div><br />
</div><br />
<br />
<br />
<h2>Select Promoter & RBS</h2><br />
<p>To select proteins expressions in wider range, you can use Promoter Selector and RBS Selector at the same time! The combination number is 320 patterns!</p><br />
<br />
<br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/Maestro_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Making various combination patarns.</span></div><br />
</div><br />
<br />
<p>Besides, you can create promoter and terminator by yourself and assemble them with our RBS Selector and GGA VECTOR (K1084301) by Golden Gate Assembly! </p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Promoter/ModelingTeam:HokkaidoU Japan/Promoter/Modeling2013-10-29T02:34:44Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Promoter</h2><br />
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<h1>Modeling</h1><br />
<br />
<p>We tried to theoretically predict the strength distribution of 4096 promoters, which were artificially created by random mutation. We followed these 3 steps, referring the previous study<sup><a href="#cite-1">[1]</a><a href="#cite-2">[2]</a></sup>.</p> <br />
<ol><br />
<li>Calculate the binding energy of each promoter and &sigma;-factor using the sequence</li><br />
<li>Convert the binding energy to the probability that RNAP binds promoter using the method of statistical mechanics</li><br />
<li>Utilizing the binding probability as the transcription efficiency</li><br />
</ol><br />
<br />
<h2>STEP 1: Calculation of Binding Energy</h2><br />
<p>First, we found the binding energy of RNAP and our promoters. As we mutated only -35 region, we only use this region for calculations. Here we define the binding energy $\varepsilon$ as the energy <span class="italic">released</span> by RNAP’s binding to promoter. Simply saying, the higher is the binding energy, the stronger is the binding. We referred the data in Kenney, <span class="italic">et al.</span><a href="#cite-3"><sup>[3]</a></sup> to calculate each binding energy.<br />
<br />
<p>The distribution of computed 4096 promoters' binding energies is shown below. The horizontal axis stands for $\varepsilon$ (at $0.05 k_BT$ intervals) and the vertical axis sample number.</p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/HokkaidoU2013_promoter_Modeling_fig1.png"><br />
<div><span class="bold">fig.1 Visualized data.</span> A portion enclosed with red square is randomized -35 region.</span></div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/1/16/HokkaidoU2013_promoter_Modeling_fig2.png"><br />
<div><span class="bold">fig.2 Promoters distribution of binding energy.</span> The result is an approximate normal distribution.</div><br />
</div><br />
<br />
<h2>STEP 2: Conversion from Binding Energy to Binding Probability</h2><br />
<br />
<br />
<p>Next, we estimated the binding probability. On this step, we used the method of statistical mechanics. So we assumed the following.</p><br />
<ul><br />
<li>The cell is a closed system</li><br />
<li>There are $P$ RNAPs bound somewhere on DNA</li><br />
<li>The number of bases is $N$ (bp) and $1$ of $N$ bases is +1 position of the promoter</li><br />
</ul><br />
<br />
<p>The principle of statistical mechanics is very easy; any state emerges with the same probability. So we counted up the number of state. A state stands for every information of all the particles in the system, so the number is enormous. $W$ represents this number. Here $W$ can be separated as the following.<br />
<br />
\[<br />
W=W_{\mathrm{unbound}}+W_{\mathrm{bound}}<br />
\]<br />
<br />
$W_{\mathrm{bound}}$ represents the number of state where the promoter is occupied and $W_{\mathrm{unbound}}$ unoccupied.</p><br />
<br />
<p>The purpose of this step is to find the ratio $W_{\mathrm{unbound}}:W_{\mathrm{bound}}$. Concerning the position of RNAP,<br />
<br />
\begin{align*}<br />
W_{\mathrm{unbound}}:W_{\mathrm{bound}}&=\frac{(N-1)!}{P!(N-P-1)!}\times W_{\mathrm{R}}(E):1 \times \frac{(N-1)!}{(P-1)!(N-P)!}\times W_{\mathrm{R}}(E+\varepsilon) \\ &=1:\frac{P}{N-P} \times \frac{W_{\mathrm{R}}(E+\varepsilon)}{W_{\mathrm{R}}(E)}<br />
\end{align*}<br />
<br />
<br />
where $W_{\mathrm{R}}$ represents the number of state in reservoir system (a system excluding the imformation of RNAP's position). $W_{\mathrm{R}}$ is a function of internal energy. Then, we converted $W_{\mathrm{R}}$ to entropy $S$ using the conversion formula: $S \equiv k_B \ln{W}$ ($k_B$ stands for Boltzmann constant, $\approx 1.38\times 10^{-23} \mathrm{J\cdot K^{-1}}$).<br />
<br />
\begin{align*}<br />
&=1:\frac{P}{N-P} \times \frac{\exp\left(\frac{S(E+\varepsilon)}{k_B}\right)}{\exp\left(\frac{S(E)}{k_B}\right)} \\ &=1:\frac{P}{N-P} \times \exp\left(\frac{S(E+\varepsilon)-S(E)}{k_B}\right) \\ &\approx 1:\frac{P}{N} \times \exp\left(\frac{\varepsilon \frac{\partial S}{\partial E}}{k_B}\right)<br />
\end{align*}<br />
<br />
Entropy $S$ and energy $E$ is connected as temperature $T$ as the following.<br />
<br />
\[<br />
\frac{\partial S}{\partial E} \equiv \frac{1}{T}<br />
\]<br />
<br />
So,<br />
<br />
\[<br />
W_{\mathrm{unbound}}:W_{\mathrm{bound}} \approx 1:\frac{P}{N} \times \exp\left(\frac{\varepsilon}{k_BT}\right)<br />
\]<br />
<br />
<br />
This is a final form of this calculation. Approximately the binding energy of -35 region is exponentially proportional to the binding probability.</p><br />
<br />
<h2>STEP 3: Conclusion</h2><br />
<p>The last step is to convert the binding probability to the transcription efficiency. Let us assume these suppositions.<br />
</p><br />
<br />
<ul><br />
<li>RNAP bound to promoter promptly initiate transcription</li><br />
<li>There is no "traffic jam" of RNAPs on DNA (i. e., RNAP's transcription initiation is rate-limiting)</li><br />
</ul><br />
<br />
<p>These assumptions mean that we can directly use the value of binding probability as transcription energy in an arbitrary unit. In this way, we get following conclusive result.</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d3/HokkaidoU2013_promoter_Modeling_fig4.png"><br />
<div><span class="bold">fig.3 Promoter distribution of transcription efficiency.</span> The horizontal axis stands for the transcription efficiency.</div><br />
</div><br />
<br />
<p>As you can see in this figure, the strengths of our promoter families vary about 1000 fold!</p><br />
<br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">Rob Phillips, Jane Kondev and Julie Theriot. (2008). <span class="italic">Physical Biology of the Cell.</span> Garland Science.</li><br />
<li id="cite-2">Brewster, <span class="italic">et al.</span> (2012). Tuning promoter strength through RNA polymerase binding site design in <span class="italic">Escherichia coli</span>. <span class="italic">PLoS computational biology.</span></li><br />
<li id="cite-3">Kenney, <span class="italic">et al.</span> (2010). Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence. <span class="italic">Proceeding of the National Academy of Sciences of the United States of America.</span></li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/PromoterTeam:HokkaidoU Japan/Promoter2013-10-29T02:32:44Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Promoter</h2><br />
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<h1>Overview</h1><br />
<p>Proteins are expressed in mainly 2 steps. First mRNA is polymerized using DNA as a template. Then ribosome binds mRNA and translates it into protein.<br />
</p><p>Promoter is a DNA sequence initiating transcription from DNA to mRNA. If transcriptional efficiency is defined as "promoter strength", stronger promoter has ability to transcribe more mRNA. This should lead in stronger expression of proteins.<br />
</p><p>We have created several promoters by randomization of -35 sequence followed by selection. In promoters -35 region is responsible for supporting binding of RNA polymerase (RNAP). This interaction results in closed complex which is rate-limiting step. We focused on this rather transparent function to introduce variability in promoter strength.<br />
</p><br />
<br />
<h2>Overview about Transcription</h2><br />
<p>We explain the importance of promoter sequence, but before that let's look how RNA binds to a promoter with the help of (fig.1).</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/igem.org/7/77/HokkaidoU_2013_Promoter_fig1.png"><br />
<div><span class="bold">fig. 1 mRNA transcription starts with promoter engagement, continues to initiation, elongation, and then it comes to termination (omitted in the figure).</span></div><br />
</div><br />
<br />
<p>First transcription complex must be formed. Transcription complex polymerizes mRNA in 2 steps. Initiation step starts polymerization followed by elongation step. Promoter serves crucial role on engagement and initiation. After closed complex formation DNA double helix pulled apart to form transcription bubble. During this closed complex changes into open complex. This marks the beginning of mRNA polymerization. Transcription bubble exposes deoxyribonucleotides to form new hydrogen bonds with ribonucleotides. In short DNA serves as template to make mRNA.</p><br />
<br />
<h2>Transcription factors related to Promtoer</h2><br />
<p>RNA complex consist of 5 core enzymes and a &sigma; factor. &sigma; factor plays crucial role in promoter recognition. It recognizes and binds to promoter region on DNA sequence and helps to assemble the core enzyme and start transcription. &sigma; factor has several analogs, <span class="italic">E. coli</span> which is widely used bacteria by iGEMers is using &sigma;70 for house-keeping gene expression at exponential growth. Bacterial promoter can be roughly divided into three regions; -10 region, spacer and -35 region. Bases in promoter are numbered in descending order from transcription start base which is defined as +1.</p><br />
<br />
<dl><br />
<dt>-10 region</dt><br />
<dd>The -10 region is structurally very important because it is initiates promoter melting in RNAP-promoter complex. This is essential to form open complex. Promoter consensus sequence is TATAAT at -12 to -7 position.</dd><br />
<br />
<dt>Spacer</dt><br />
<dd>Spacer is thought to increase flexibility of &sigma; factor binding requirements.</dd><br />
<br />
<dt>-35 region</dt><br />
<dd>-35 region is second in importance to -10. It does not energetically contribute to promoter melting. There reports on promoters without -35 region. In those case TG motif at about -16 is thought as alternative. -35 consensus sequence is TTGACA at from -36 to -31.</dd><br />
</dl><br />
<br />
<p>Promoters function to bind RNAP is a reason it is genetically well preserved. Most frequently conserved residues in the sequence make a "consensus sequence". In 1983, -35 and -10 consensus was showed to be TTGACA and TATAAT respectively (fig 2). Horizontal axis of the figures represents the position upstream of translation ignition point. Letter at the top of the figure signifies more than over 39% occurrence of that letter at that position. Larger occurrence over 54% is represented as upper case letter. Consensus sequence published by Marjan De Mey <span class="italic">et al</span>. (2007) shows that -10 and -35 region is highly preserved (fig 3). There other less preserved regions. The tetramer (TRTG) upstream from -10 region is called TG motif. Upstream of -35 region is UP element and downstream of -10 region is discriminator region. These sequences are thought to bind core enzymes. So these sequences are also well conserved. Each sequence is important to control promoter strength.</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d6/HokkaidoU2013_Promoter_background_fig3_new_800.png"><br />
<div><span class="bold">fig. 2 Consensus sequence shown in review article in 1983 [3].</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_promoter_Background_fig4.png"><br />
<div><span class="bold">fig. 3 Consensus sequence prepared in 2007 [4].</span></div><br />
</div><br />
<br />
<br />
<p>So we went and designed "consensus promoter". It should have strongest binding energy to RNAP. By adding mutations to -35 we sought to construct promoters with various binding energies. There are three reasons why we used -35 region.<br />
</p><p>First, -35 region is just supporting binding with &sigma; factor. It has less vital role compared to -10 region, which energetically contributes to formation of open complex. Having this in mind we changed -35 region to easily change promoter binding strength without severe errors in promoter function.<br />
</p><p>Second, RNAP and promoter binding orchestrated by &sigma; factor binding. Complex formation is thought to be rate-limited step. We thought that -35 region performs a simpler function. For this reason, mutations at -35 region can be thought as more structurally transparent.<br />
</p><p>Recently published research reported the making of promoter family by randomizing both -35 and -10 regions, changing spacer length. However it would be too much of the task for us to make some many changes. By changing hexamer sequence of -35 region there are 4096 variation. This number is a lot smaller compared to mutating every promoter position. So we can get result with a smaller library size.<br />
</p><p>With these 3 reasons we went on to construct our promoter family.<br />
</p><br />
<br />
<p><br />
<li><br />
[1] R. a Mooney, I. Artsimovitch, and R. Landick, “Information processing by RNA polymerase: recognition of regulatory signals during RNA chain elongation.,” Journal of bacteriology, vol. 180, no. 13, pp. 3265–75, Jul. 1998.<br />
</li><br />
<li><br />
[2] M. S. B. Paget and J. D. Helmann, “The σ 70 family of sigma factors,” Genome Biology, vol. 4, no. 1, pp. 203.1–203.6, 2003.<br />
</li><br />
<li><br />
[3] D. K. Hawley, W. R. Mcclure, and I. R. L. P. Limited, “Compilation and analysis of <span class="italic">Escherichia coli</span> promoter DNA sequences,” <br />
</li><br />
Nucleic Acids Research, vol. 11, pp. 2237–2255, 1983.<br />
<li><br />
[4] M. De Mey, J. Maertens, G. J. Lequeux, W. K. Soetaert, and E. J. Vandamme, “Construction and model-based analysis of a promoter library for <span class="italic">E. coli</span>: an indispensable tool for metabolic engineering.,” BMC biotechnology, vol. 7, p. 34, Jan. 2007. <br />
</li><br />
<li><br />
[5]De Mey, M., Maertens, J., Lequeux, G. J., Soetaert, W. K., & Vandamme, E. J. (2007). Construction and model-based analysis of a promoter library for <span class="italic">E. coli</span>: an indispensable tool for metabolic engineering. BMC biotechnology, 7, 34. doi:10.1186/1472-6750-7-34<br />
</li><br />
</p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_WorkTeam:HokkaidoU Japan/Shuffling Kit/Future Work2013-10-29T02:30:45Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E. coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Maestro E.coli Future Work</h1><br />
<br />
<h2>To make Promoter Selector better kit</h2><br />
<p>To make Promoter Selector better, we are going to place pigment producing construct between two PstI sites. After you confirm the best promoter, you can remove the followed constructs by digesting with PstI.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/12/Fig11_new2_800_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 How to make our kit better.</span></div><br />
</div><br />
<br />
<h2>Application of RBS Selector</h2><br />
<p>We can get a lot of operons by RBS Selector. RBS Selector provides 16 operons in case of 2 CDS, 64 operons in case of 3 CDS. What can we do using these number of operons? We expect industrial applications of RBS Selector as future works.</p><br />
<br />
<p><br />
In the case of producing valuable molecules inside the cell, it is important to select the RBS which results the largest yield.<br />
<br />
<br />
For example PHB <br />
(polyhydroxybutyrate) ,3 enzymes synthesize PHB from substrate. PHB is one of the eco-friendly, biodegradable plastic. You can learn more about PHB at <a href="https://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis">iGEM HokkaidoU_Japan 2012 wiki</a>. In the production of PHB, each CDS has the optimum RBS. Our RS can be used for selecting the best combination of RBSs.<br />
<br />
<br />
<br />
It is possible to use RBS Selector when producing medicine or vaccine etc.<br />
<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2012/3/38/HokkaidoU_PHB_Fig3.jpg"><br />
<div><span class="bold">fig.2 P(3HB) synthesis pathway in R. eutropha.</span></div><br />
</div><br />
<br />
<br />
<h2>Select Promoter & RBS</h2><br />
<p>To select proteins expressions in wider range, you can use Promoter Selector and RBS Selector at the same time! The combination number is 320 patterns!</p><br />
<br />
<br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/Maestro_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Making various combination patarns.</span></div><br />
</div><br />
<br />
<p>Besides, you can create promoter and terminator by yourself and assemble them with our RBS Selector and GGA VECTOR (K1084301) by Golden Gate Assembly! </p><br />
<div class="clearfix"></div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Overview/BackgroundTeam:HokkaidoU Japan/Overview/Background2013-10-29T02:27:19Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Overview</h2><br />
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<h1>Background</h1><br />
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<p><br />
Synthetic biology is one of the most interesting fields in 21st century.<br />
Its goal is to comprehend and reproduce the marvels of living things.<br />
As members of syn-bio community, a lot of iGEMers have found interesting proteins.<br />
However, the focuses of these projects are “qualities” of proteins.<br />
As well as the uniqueness of proteins, the expression levels of proteins contribute to the marvelous functions of living things.<br />
The next goal of syn-bio is to control “quantities” of proteins.<br />
</p><br />
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<p><br />
To control protein quantities in the organism, we must control transcription, a step from DNA to mRNA and translation, a step from mRNA to protein.<br />
Transcription is regulated by promoter region.<br />
Translation is regulated by ribosome binding site: RBS.<br />
Depending on promoters, the transcription rate varies about 1000 fold.<br />
Similarly, depending on RBSs, the translation rate about 100 fold.<br />
So, to control quantity, it is essential to figure out the characteristics of these expression-regulatory regions and adjust them.<br />
</p><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8a/HokkaidoU2013_Motivation_figure4_new.png"><br />
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<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU2013_Motivation_figure5.png"><br />
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<p><br />
Recently, many studies have been done to theoretically predict the amount of gene expression by the sequences of expression-regulatory regions.<br />
There are some accurate <span class="italic">in vivo</span> expression efficiency predictions.<br />
However, even if you were able to predict it exactly, you could not adjust it to an optimal level because you don't know the best rate for the bacteria.<br />
</p><br />
<br />
<p><br />
Suppose you are planning to express beneficial product, like antibodies.<br />
You may think that the strongest promoter and RBS lead the maximal yield of the proteins.<br />
But actually, selecting the strongest one isn't always the best strategy.<br />
Overexpressed proteins can be perceived as detrimental by bacterial immune system and packed into inclusion bodies.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_Motivation_figure1.png"><br />
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<p><br />
Another example is a multiple expression, such as mixing color of chromoprotein.<br />
Theoretically, three different chromoproteins can express any kind of colors by fixing each expression level.<br />
But this adjustment is too sensitive to be made instinctively.<br />
</p><br />
<p><br />
Furthermore, the fact that sequence of CDS may affect transcription or translation efficiency is known from some studies.<br />
It means the strength of promoter or RBS could be changed by CDS.<br />
</p><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/7/76/HokkaidoU2013_Motivation_figure3_big.png"><br />
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<p><br />
Over these challenging problems in syn-bio, we put forward one solution like a Columbus's egg.<br />
"No more prediction, let's experiment!" ---- that is, we should try several promoters or RBSs and select the best one or the best combination.<br />
We don't know the best answer, but bacteria do.<br />
</p><br />
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<br />
<p><br />
This is surely the best way, but trying every pattern costs a lot of time and labor.<br />
We overcame this obstacle by using a one-pot DNA shuffling method, namely, "Golden Gate Assembly".<br />
And then, we made two kits to optimize transcription and translation.<br />
For these kits, we made artificial promoter and RBS families.<br />
They are created based on concrete philosophies and characterized well.<br />
Please refer <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Promoter">Promoter</a> and <a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS">RBS</a> for details.<br />
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<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/HokkaidoU2013_Motivation_figure2.png"><br />
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<p><br />
We named these kits "Promoter Selector" and "RBS Selector".<br />
<dl><br />
<dt>Promoter Selector</dt><dd>Promoter Selector is for adjustment of relatively simple expression, like a production of antibodies reffered above.</dd><br />
<dt>RBS Selector</dt><dd>RBS Selector is for adjustment of multiple expressions, like complex metabolisms.</dd><br />
</dl><br />
We named these two devices ''Maestro <span class="italic">E. coli</span>'' Random Operon Shuffling Kit! As a maestro harmonizes music, let's harmonize proteins in the bacteria! <br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Future_WorkTeam:HokkaidoU Japan/Shuffling Kit/Future Work2013-10-28T19:57:02Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Maestro E.coli Future Work</h1><br />
<br />
<h2>To make Promoter Selector better kit</h2><br />
<p>To make Promoter Selector better, we are going to place pigment producing construct between two PstI sites. After you confirm the best promoter, you can remove pigment producing constructs by digesting with PstI.</p><br />
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<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/12/Fig11_new2_800_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 How to make our kit better.</span></div><br />
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<h2>Application of RBS Selector</h2><br />
<p>We can get a lot of operons by RBS Selector. RBS Selector provides 16 operons in case of 2 CDS, 64 operons in case of 3 CDS. What can we do using these number of operons? We expect industrial applications of RBS Selector as future works.</p><br />
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<p><br />
In the case of producing valuable molecules inside the cell, it is important to select the RBS which results the largest yield.<br />
<br />
<br />
For example PHB <br />
(polyhydroxybutyrate) ,3 enzymes synthesize PHB from substrate. PHB is one of the eco-friendly, biodegradable plastic. You can learn more about PHB at <a href="https://2012.igem.org/Team:HokkaidoU_Japan/Project/PHB_Synthesis">iGEM HokkaidoU_Japan 2012 wiki</a>. In the production of PHB, each CDS has the optimum RBS. Our RS can be used for selecting the best combination of RBSs.<br />
<br />
<br />
<br />
It is possible to use RBS Selector when producing medicine or vaccine etc.<br />
<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2012/3/38/HokkaidoU_PHB_Fig3.jpg"><br />
<div><span class="bold">fig.2 P(3HB) synthesis pathway in R. eutropha.</span></div><br />
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<h2>Select Promoter & RBS</h2><br />
<p>To select proteins expressions in wider range, you can use Promoter Selector and RBS Selector at the same time! The combination number is 320 patterns!</p><br />
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<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3b/Maestro_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Making various combination patarns.</span></div><br />
</div><br />
<br />
<p>Besides, you can create promoter and terminator by yourself and assemble them with our RBS Selector and GGA VECTOR (K1084301) by Golden Gate Assembly! </p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/ExamplesTeam:HokkaidoU Japan/Shuffling Kit/Examples2013-10-28T19:56:42Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<h1>Demonstrations for Usecase Example</h1><br />
<p>We will show some interesting demonstrations of our kits, Promoter Selector and RBS Selector!</p><br />
<br />
<br />
<h2>Promoter Selector</h2><br />
<p>Let's select the best promoter for Kanamycin resistance by Promoter Selector.</p><br />
<p>For a demonstration we decided to optimize the expression of Kanamycin resistance. Changing the concentration of Kanamycin in agar plate, it is estimated that different promoter will be chosen by our Promoter Selector (fig.1).</p><br />
<br />
<p>If the concentration of Kanamycin was high, the colony with strong promoter will survive. Therefore, only one or two colors indicate the first and second biggest occupancy rate on the plate.<br />
If the concentration of Kanamycin was low, colonies with weak promoters will be able to survive. This way many colors of colonies would appear (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bb/Fig1_in_example_132029new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 Different promoter express each colors.</span></div><br />
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<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/Fig2_in_example_HokkaidoU_2013.png"><br />
<div style="padding-bottom: 0;"><span class="bold">fig.2 Difference of Kanamycin concentration.</span></div><br />
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<h4>Method</h4><br />
<br />
<p>Optimum concentration of Kanamycin: in LB is 50 mg/ml<br />
We prepared 3 different concentration plates. <br />
</p><br />
<br />
<ul><br />
<li>Plate A: Kanamycin 125 mg per plate</li><br />
<li>Plate B: Kanamycin 250 mg per plate</li><br />
<li>Plate C: Kanamycin 500 mg per plate (optimum concentration)</li><br />
<li>Plate D: Kanamycin 1000 mg per plate</li><br />
</ul><br />
<br />
<p>Gene<br />
Vector: pSB1C3<br />
</p><br />
<p>We cloned Kanamycin resistant gene from pSB3K3, by using BsaI adding primer. Used the Promoter Selector (K1084501, K1084502, K1084503, K1084504, K1084505 ).</p><br />
<br />
<p><br />
Culture: 37 &deg;C, for 48h<br />
</p><br />
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<h4>Results</h4><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU_2013_Km_resistance_assay_summary_data.png"><br />
<div><span class="bold">fig.4 Graph of number and rate, and table of number of colonies size over 1mm diameter.</span></div><br />
</div><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/c/ca/POK_DEMO_48h_newnew_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.3 Picture of plate B (Kanamycine 250 mg). The colonies showed several colors.</span></div><br />
</div><br />
<br />
<p>After 48h cultivation, around 300 colonies had appeared on each LBKC (Kanamycin and Chloramphenicol) plates. We prepared LacZa expression in Promoter Selector system as negative control to estimate the success of Golden Gate Assembly, and only 7 to 0 colonies are expressed LacZa. Mixed colored colonies which would have been transformed by two or more Promoter Selector were also observed. The number and rate of colonies per each plate were graphed (fig.4), with rejecting these undesirable colonies.<br />
</p><br />
<br />
<p><br />
In (fig.4), legend color corresponds to Promoter Selector’s part number. The sum of colony numbers is displayed above each bar, and rate is in these sections. Number in the table is the number of each Promoter Selector’s colonies. These data are collected from only one time Kanamycin resistance assay result.<br />
</p><br />
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<div class="clearfix"></div><br />
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<h4>Conclusion</h4><br />
<p><br />
There is no difference from lowest and highest Kanamycin concentration. In these colonies, number of colonies derived from K1084405 (containing K1084010 promoter ) has the most largest rate on each plate. This result suggests that the colonies expressed the lowest amount of Kanamycin resistance gene, and the resorce of transcription and translation could be spared to cell growth,thus the number of colonies may have been largest. Otherwise, the DNA solution of K1084505 Promoter Selector used at ligation was simply larger than other DNA solution. Although the result is collected from only one time assay, higher conscentration of Kanamycin and much number of trials than this time will be needed.<br />
</p><br />
<br />
<p><br />
From these result and the experimental fact, the existence of Km resistance gene in Promoter Selector’s BsaI cloning section is partially confirmed. Our Promoter Selector was successfully assembled, but it does not adopted to all colonies. Then, as a result of assembling, we succeeded in making colorful colonies appear on one plate.<br />
</p><br />
<h2>RBS Selector</h2><br />
<h3>4 colors</h3><br />
<p>Let’s create all combinations by two reporter genes and make various colors on one plate!</p><br />
<br />
<br />
<p><br />
The RBS Selector we made, can randomize the strength of RBSs in the operon.<br />
For a demonstration, we decided to create all combinations by two genes; mRFP1 (BBa_E1010) and LacZ&alpha; (BBa_I732006) (fig.5). LacZ&alpha; makes the colony blue. mRFP1 makes the colony red.<br />
</p><br />
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<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/90/Fig4_in_example_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Create all combinations by RBS of defferent stlength mRFP1 (BBa_E1010) and LacZα (BBa_I732006).</span></div><br />
</div><br />
<br />
<p>When the RBS upstream of mRFP1 was strong and the RBS upstream was weak, the colony should be red. When the RBS upstream of mRFP1 was weak, and the RBS upstream was strong, the colony should be blue. So when if the strength of RBS upstream both genes were the same, colony will be white, purple (fig.6).</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c8/Fig5_in_example_%2Boverhang_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Each combinations of RBS make different colors.</span></div><br />
</div><br />
<br />
<h4>Method</h4><br />
<ul><br />
<li>Used promoter1 (BBa_K1084001), SD2 (BBa_K1084101), SD4 (BBa_K1084102) and assembled with.</li><br />
<li>Spread X-GAL(250 mg)on LBC plate.</li><br />
<li>Cultured for 37 &deg;C, 26h.</li><br />
</ul><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/0/08/ROK_demo_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The colonies showed red, blue, white, and purple.</span></div><br />
</div><br />
<h4>Results</h4><br />
<p><br />
We got many colored colonies,red, blue, white, and purple.<br />
<br />
<br />
</p><br />
<div class="clearfix"></div><br />
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<h4>Conclusion</h4><br />
<br />
<br />
<p><br />
<br />
We can say that our RBS Selector worked!!<br />
The RBSs uptsream 2 genes were randomized and they had many levels of expressions. <br />
<br />
</p><br />
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<h3>64 colors</h3><br />
<br />
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<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/64demo2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.8.</span></div><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
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<h1>Primer Designer for Maestro</h1><br />
<br />
<h3>1. First, you have to decide whether or not you conform to our default overhang set.</h3><br />
<div class="radio"><br />
<label><br />
<input type="radio" name="default" value="1">Yes, I conform to the default.</input><br />
</label><br />
</div><br />
<div class="radio"><br />
<label><br />
<input type="radio" name="default" value="2">No, I design original overhangs.</input><br />
</label><br />
</div><br />
<br />
<div id="conform"><br />
<h3>2. Secondly, you hove to determine how many CDS are included in your target plasmid.</h3><br />
I have<br />
<select class="form-control" name="cds-number"><br />
<option value="1">1</option><br />
<option value="2">2</option><br />
<option value="3">3</option><br />
</select><br />
CDS(s) included.<br />
<br />
<h3>3. Then, please select the region to where your fragment correspond in target plasmid.</h3><br />
<div id="plasmid-map"><br />
<div id="plasmid-0"> </div><br />
<div class="plasmid-gray" id="plasmid-1"> <span id="indicator1">1</span> </div><br />
<div class="plasmid-gray" id="plasmid-2"> <span id="indicator2">2</span> </div><br />
<div class="plasmid-gray" id="plasmid-3"> <span id="indicator3">3</span> </div><br />
<div class="plasmid-gray hide-1" id="plasmid-4"> <span id="indicator4">4</span> </div><br />
<div class="plasmid-gray hide-1" id="plasmid-5"> <span id="indicator5">5</span> </div><br />
<div class="plasmid-gray hide-2" id="plasmid-6"> <span id="indicator6">6</span> </div><br />
<div class="plasmid-gray hide-2" id="plasmid-7"> <span id="indicator7">7</span> </div><br />
<div class="plasmid-gray" id="plasmid-8"> <span id="indicator8">8</span> </div><br />
<div id="plasmid-9"> <span id="indicator9">9</span> </div><br />
</div><br />
<br />
<div class="my-form"><br />
<div class="form-group"><br />
<label for="part-beginning"><br />
Beginning of the fragment:<br />
</label><br />
<select name="part-beginning" class="form-control monospace"><br />
<option value="1">1: CGTC</option><br />
<option value="2">2: AAGG</option><br />
<option value="3">3: CTGA</option><br />
<option class="disable-1" value="4">4: TTAT</option><br />
<option class="disable-1" value="5">5: TTCG</option><br />
<option class="disable-2" value="6">6: TAGA</option><br />
<option class="disable-2" value="7">7: TCCC</option><br />
<option value="8">8: CGGT</option><br />
<option value="9">9: AGTA</option><br />
</select><br />
</div><br />
<div class="form-group"><br />
<label for="part-end"><br />
End of the fragment:<br />
</label><br />
<select name="part-end" class="form-control monospace"><br />
<option value="1">1: CGTC</option><br />
<option value="2">2: AAGG</option><br />
<option value="3">3: CTGA</option><br />
<option class="disable-1" value="4">4: TTAT</option><br />
<option class="disable-1" value="5">5: TTCG</option><br />
<option class="disable-2" value="6">6: TAGA</option><br />
<option class="disable-2" value="7">7: TCCC</option><br />
<option value="8">8: CGGT</option><br />
<option value="9">9: AGTA</option><br />
</select><br />
</div><br />
</div><br />
<br />
<br />
<h3>4. Okey, now we are ready to go. Enter your fragment's sequence, and press "Calculate"!</h3><br />
</div><br />
<br />
<div id="not-conform"><br />
<h3>2. Please enter overhangs and fragment's sequence, and press "Calculate"</h3><br />
</div><br />
<br />
<form id="primer-designer" class="my-form"><br />
<div class="form-group"><br />
<label for="overhang-f"><br />
Forward primer overhang:<br />
</label><br />
<input class="form-control monospace" type="text" name="overhang-f"><br><br />
</div><br />
<div class="form-group"><br />
<label for="overhang-r"><br />
Reverse primer overhang:<br />
</label><br />
<input class="form-control monospace" type="text" name="overhang-r"><br><br />
</div><br />
<div class="form-group"><br />
<label for="sequence"><br />
Fragment sequence:<br />
</label><br />
<textarea class="form-control monospace" type="text" name="sequence"></textarea><br><br />
</div><br />
<div class="form-group"><br />
<label></label><br />
<button class="form-control">Calculate</button><br />
</div><br />
</form><br />
<br />
<hr style="margin: 50px 0;"><br />
<br />
<h4>Forward</h4><br />
<dl><br />
<dt>sequence</dt><br />
<dd><pre id="primer-f" class="monospace"></pre></dd><br />
<dt>tm</dt><br />
<dd><pre id="tm-f"></pre></dd><br />
</dl><br />
<h4>Reverse</h4><br />
<dl><br />
<dt>sequence</dt><br />
<dd><pre id="primer-r" class="monospace"></pre></dd><br />
<dt>tm</dt><br />
<dd><pre id="tm-r"></pre></dd><br />
</dl><br />
<br />
<h3>5. Now, repeat previous step for remaining fragments included in the target plasmid.</h3><br />
<script type="text/javascript"><br />
(function(){var n,f,e,h,d,a,j,g,i,b,m,k,c,l;a=function(o){switch(o){case"AA":case"TT":return -9.1;case"AT":return -8.6;case"TA":return -6;case"CA":case"TG":return -5.8;case"GT":case"AC":return -6.5;case"CT":case"AG":return -7.8;case"GA":case"TC":return -5.6;case"CG":return -11.9;case"GC":return -11.1;case"GG":case"CC":return -11}};k=function(o){var p,q,s,r;q=0;for(p=s=0,r=o.length-2;0<=r?s<=r:s>=r;p=0<=r?++s:--s){q+=a(o.slice(p,+(p+1)+1||9000000000))}return q};j=function(o){switch(o){case"AA":case"TT":return -24;case"AT":return -23.9;case"TA":return -16.9;case"CA":case"TG":return -12.9;case"GT":case"AC":return -17.3;case"CT":case"AG":return -20.8;case"GA":case"TC":return -13.5;case"CG":return -27.8;case"GC":return -26.7;case"GG":case"CC":return -26.6}};c=function(o){var p,q,s,r;q=0;for(p=s=0,r=o.length-2;0<=r?s<=r:s>=r;p=0<=r?++s:--s){q+=j(o.slice(p,+(p+1)+1||9000000000))}return q};n=function(o){var q,p;q=k(o);p=c(o);return(1000*q/(-10.8+p+1.987*-15.89495209964411))-273.15+16.6*-1.3010299956639813};d=function(p){var r,o,q;if(n(p.slice(0,35))<60){alert("Sequence is too short.");return false}for(r=q=17;q<=35;r=++q){o=n(p.slice(0,+(r-1)+1||9000000000));if(o>60){if(p[r-1]==="G"||p[r-1]==="C"){break}}}return[p.slice(0,+(r-1)+1||9000000000),o]};h=function(o){switch(o){case"A":return"T";case"T":return"A";case"G":return"C";case"C":return"G"}};m=function(p){var o;return((function(){var t,r,s,q;s=p.split("");q=[];for(t=0,r=s.length;t<r;t++){o=s[t];q.push(h(o))}return q})()).reverse().join("")};g=function(s,o,w){var t,p,r,u,v,q;t=d(w);p=d(m(w));r="TTTGGTCTCT"+s+"T"+t[0];v=t[1];u="TTTGGTCTCA"+o+"A"+p[0];q=p[1];return[r,v,u,q]};e=function(p){var o;o=true;if(/GGTCTC/.test(p)||/GGTCTC/.test(m(p))){alert('This sequence contains BsaI cutting site "GGTCTC" or "GAGACC".\r\nYou can just ignore this alert, if unnecessary.');o=false}if(/GAATTC/.test(p)||/GAATTC/.test(m(p))){alert('This sequence contains EcoRI cutting site "GAATTC".\r\nYou can just ignore this alert, if unnecessary.');o=false}if(/CTGCAG/.test(p)||/CTGCAG/.test(m(p))){alert('This sequence contains PstI cutting site "CTGCAG".\r\nYou can just ignore this alert, if unnecessary.');o=false}if(/GCGGCCGC/.test(p)||/GCGGCCGC/.test(m(p))){alert('This sequence contains NotI cutting site "GCGGCCGC".\r\nYou can just ignore this alert, if unnecessary.');o=false}if(/ACTAGT/.test(p)||/ACTAGT/.test(m(p))){alert('This sequence contains SpeI cutting site "ACTAGT".\r\nYou can just ignore this alert, if unnecessary.');o=false}if(/TCTAGA/.test(p)||/TCTAGA/.test(m(p))){alert('This sequence contains XbaI cutting site "TCTAGA".\r\nYou can just ignore this alert, if unnecessary.');o=false}return o};f=function(p){var o;o=/[ATCG]+/.exec(p);if(o[0]===p){return true}else{return false}};b=function(p){var o;o=p.val().toUpperCase().split(/[\s\n\r]+/).join("");if(!f(o)){alert("You can NOT use non-AGCT characters.");return false}if(!e(o)){return false}};i=function(o){switch(o){case 1:return"CGTC";case 2:return"AAGG";case 3:return"CTGA";case 4:return"TTAT";case 5:return"TTCG";case 6:return"TAGA";case 7:return"TCCC";case 8:return"CGGT";case 9:return"AGTA"}};l=function(){var t,o,p,s,r,q;t=parseInt($('[name="part-beginning"]').val());o=parseInt($('[name="part-end"]').val());for(p=s=1;1<=t?s<t:s>t;p=1<=t?++s:--s){$("#plasmid-"+p).addClass("plasmid-gray")}for(p=r=t;t<=9?r<=9:r>=9;p=t<=9?++r:--r){$("#plasmid-"+p).removeClass("plasmid-gray")}for(p=q=o;o<=9?q<=9:q>=9;p=o<=9?++q:--q){$("#plasmid-"+p).addClass("plasmid-gray")}if(o>t){$('[name="overhang-f"]').val(i(t));return $('[name="overhang-r"]').val(i(o))}else{$('[name="overhang-f"]').val("");return $('[name="overhang-r"]').val("")}};$(function(){$('[name="overhang-f"]').focusout(function(){return b($(this))});$('[name="overhang-r"]').focusout(function(){return b($(this))});$('[name="sequence"]').focusout(function(){return b($(this))});$("#primer-designer").submit(function(r){var q,p,u,s,o,t;r.preventDefault();p=$(this);q=p.find("button");q.attr("disabled",true);u=p.find('[name="overhang-f"]').val().toUpperCase();s=p.find('[name="overhang-r"]').val().toUpperCase();t=p.find('[name="sequence"]').val().toUpperCase().split(/[\s\n\r]+/).join("");o=g(u,m(s),t);$("#primer-f").text(o[0]);$("#tm-f").html(""+(o[1].toFixed(2))+" &deg;C");$("#primer-r").text(o[2]);$("#tm-r").html(""+(o[3].toFixed(2))+" &deg;C");return q.attr("disabled",false)});$("#not-conform").hide();$('input[name="default"]:radio').change(function(){switch($(this).val()){case"1":$("#conform").show();return $("#not-conform").hide();case"2":$("#conform").hide();return $("#not-conform").show()}});$('[name="cds-number"]').val("3");$('[name="cds-number"]').change(function(){switch($(this).val()){case"1":$(".hide-1").hide();$(".hide-2").hide();$(".disable-1").attr("disabled","disabled");return $(".disable-2").attr("disabled","disabled");case"2":$(".hide-1").show();$(".hide-2").hide();$(".disable-1").removeAttr("disabled");return $(".disable-2").attr("disabled","disabled");case"3":$(".hide-1").show();$(".hide-2").show();$(".disable-1").removeAttr("disabled");return $(".disable-2").removeAttr("disabled")}});$('[name="part-beginning"]').change(function(){return l()});return $('[name="part-end"]').change(function(){return l()})})}).call(this);<br />
</script><br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_Use"><div class="arrow-div"></div><span>How To Use</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Examples"><div class="arrow-div"></div><span>Examples</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_KitTeam:HokkaidoU Japan/Shuffling Kit2013-10-28T19:55:51Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>Overview </h1><br />
<p><br />
As we referred at the motivation, the transcription efficiency can vary about 1000 fold by choosing different promoter. Translation efficiency varies about 100 folds by choosing different RBS. So the selection of the expression regulatory region is extremely important for protein you want to express.<br />
</p><br />
<p><br />
However, selecting the strongest regulatory region does not always result in the best production. It is a hard work to select the best combination of a promoter and a RBS by yourself.<br />
</p><br />
<p><br />
Therefore, we made a remarkable kit to select the regulatory region and optimize the expression, which suits the users demand and can be done in single pot reaction. It will be a new standard of choosing promoters and RBSs!<br />
</p><br />
<p><br />
Our kits are for both promoters and RBSs. Both kits use Golden Gate Assembly (GGA), one-pot DNA shuffling (Engler, 2009). BsaI cloning site used by that method is the key of our kits.<br />
</p><br />
<p><br />
We made a animation movie to introduce our project.<br><br />
<div style="text-align: center; margin-top: 20px;"><iframe width="854" height="480" src="//www.youtube.com/embed/N0YjklSmyN0?rel=0" frameborder="0" allowfullscreen></iframe></div><br />
</p><br />
<br />
<h2>One-pot DNA shuffling method</h2><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/6/6f/HokkaidoU2013_optimization_Fig1.png"><br />
<div>fig.1 <span class="bold">BsaI cloning site.&nbsp;</span> BsaI recognizes GGTCTC sequence, and cuts other region.</div><br />
</div><br />
<p><br />
BsaI cloning site has unique characteristics that enabled us to construct our optimization kit. BsaI restriction enzyme is classified as Type IIs restriction endonuclease. The unique property of this class is that recognition site and the cutting sites are apart. Unlike EcoRI, BsaI recognizes GGTCTC sequence, but cuts the sequence located 7 bases downstream from first base in recognition site (fig.1). Which results in a 5 prime 4 base overhang structure (fig.2).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/95/HokkaidoU2013_optimization_Fig2_ver2_800.png"><br />
<div><br />
fig.2 <span class="bold">How BsaI works.&nbsp;</span> BsaI cuts different region form recognition site.<br />
</div><br />
</div><br />
<p><br />
Usual restriction enzyme has a decided overhang sequence, and the sequence is palindromic. So DNA cloning requires more than 1 kind of restriction enzyme. In contrast, BsaI DNA overhangs can be made into <span class="italic">any desired</span> sequence. Therefore, 256 different overhangs can be created using a BsaI restriction site. By designing the overhangs, DNA fragments can be assembled together in a defined order, and inserted in a vector in one step.<br />
</p><br />
<br />
<h2>Shuffling promoters and RBSs by using GGA</h2><br />
<p><br />
Our kit is using this remarkable method called "Golden Gate Assembly one-pot DNA shuffling". Thus, our kit is capable to make several variants of the constructs in one reaction.<br />
</p><br />
<p><br />
Promoter Selector can insert protein sequence into constructs with five different promoters with different strengths (fig.3). <br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7c/HokkaidoU2013_optimization_Fig3_ver2_800.png"><br />
<div><br />
fig.3 <span class="bold">Promoter shuffling.&nbsp;</span> Your CDS will be inserted the downstream of a certain promoter randomly.<br />
</div><br />
</div><br />
<p><br />
RBS Selector can insert four different RBS with different strengths. Currently it is possible to shuffle up to 3 RBSs in one operon with our kit (fig.4).<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cb/HokkaidoU2013_optimization_Fig4_800.png"><br />
<div>fig.4 <span class="bold">RBS shuffling.&nbsp;</span> RBS Selector contains 4RBSs, and you can randomize max 3 CDS.</div><br />
</div><br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_Use"><div class="arrow-div"></div><span>How To Use</span></a><br />
</div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_UseTeam:HokkaidoU Japan/Shuffling Kit/How To Use2013-10-28T19:53:42Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Shuffling Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>How to use</h1><br />
<h2>What users should prepare</h2><br />
<p><br />
To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"Primer Designer for Maestro"!</a><br />
</p><br />
<br />
<h2>Promoter Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.<br />
</p><br />
<div class="fig fig800"><br />
<style type="text/css"><br />
table color expression construct downstream of protein insertion site { border: 1px solid #a4a4a4; margin: 0 auto; }<br />
td, th { text-align: center; }<br />
</style><br />
<table><br />
<tr><br />
<th>Part number</th><th>Promoter</th><th>Promoter strength</th><th>Paired protein</th><th>Protein color</th><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084005">BBa_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td><br />
</tr><br />
</table><br />
<br />
<div><span class="bold">table.1 Matching list of promoters and their colors.</span></div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/6/60/Fig1_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.1 The matching color for respective promoters.</span></div><br />
</div><br />
<p><br />
The color always expresses. LacZ&alpha; reporter is placed between two BsaI sites (fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cc/Fig.2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.2 Insertion of LacZα reporter between two BsaI sites.</span></div><br />
</div><br />
<br />
<h2>How it works</h2><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png"><br />
<div><span class="bold">fig.3 Digestion by BsaI.</span></div><br />
</div><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should do the trick.<br />
</p><br />
<br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)<sup><a href="#cite-1">[1]</a></sup>. All the protein coding sequence will be inserted in the plasmid.<br />
<!-- add more detail --><br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/Fig4_131027_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.4 How to insert CDS into protein coding sequence.</span></div><br />
</div><br />
<br />
<p><br />
3. You should get the construct shown below after Golden Gate Assembly (fig.5).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a1/Fig5_131027_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.5 Constructs you can get.</span></div><br />
</div><br />
<p><br />
4. Transform the ligated DNA to <span class="italic">E. coli</span>, and spread it on plate. Then, you will get colonies with five colors easily because you can see 5 colors by naked eyes (fig.6). The colors are paired with the promoters, so you will know what promoter you are using without sequencing all the colonies. You can pick up colonies and have an assay.</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/0/07/Assay_new_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.6 Method after transformation.</span></div><br />
</div><br />
<p><br />
<br />
<br />
<h2>RBS Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our kit contains tandem RBS (fig.7) and acceptor plasmid (fig.8).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/22/RBS_strength_2_HokkaidoU_2013.png"><br />
<div><span class="bold">fig.7 The sequence of tandem RBS.</span></div><br />
</div><br />
<p><br />
In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs are connected together.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png"><br />
<div><span class="bold">fig.8 The acceptor part of the RBS and protein coding region.</span> It has a BsaI site for the parts to be assembled.</div><br />
</div><br />
</p><br />
<br />
<h3>How to use</h3><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence. Again, <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a> should help your primers design (fig.9). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png"><br />
<div><span class="bold">fig.9 Insertion CDS anf digestion by BsaI.</span></div><br />
</div><br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.10).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png"><br />
<div><span class="bold">fig.10 How to use Golden Gate Assembly.</span></div><br />
</div><br />
<br />
<p><br />
3. Transform the ligated DNA to <span class="italic">E. coli</span>. If you are optimizing three different proteins, you will get 64 different kinds of constructs.<br />
</p><br />
<br />
<p><br />
We will submit these standard methods as RFC to BioBrick Foundation.<br />
</p><br />
<br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">C. Engler <span class="italic">et al.</span> Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE</li><br />
</ol><br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit"><div class="arrow-div"></div><span>Shuffling Kit Top</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ResultsTeam:HokkaidoU Japan/RBS/Results2013-10-28T16:09:16Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Results</h1><br />
<p><br />
We made these constructs to measure translation efficiency of our RBSs. We assembled LacZ&alpha; to performed &beta;-Galactosidase assay.<br />
</p><br />
<dl><br />
<dt>negative control</dt><dd>No insert</dd><br />
<dt>positive control</dt><dd>pTet-B0034-LacZ&alpha;-dT</dd> <br />
<dt>SD2</dt><dd>pTet-SD2-LacZ&alpha;-dT (BBa_K1084121)</dd><br />
<dt>SD4</dt><dd>pTet-SD4-LacZ&alpha;-dT (BBa_K1084122)</dd><br />
<dt>SD6</dt><dd>pTet-SD6-LacZ&alpha;-dT (BBa_K1084123)</dd><br />
<dt>SD8</dt><dd>pTet-SD8-LacZ&alpha;-dT (BBa_K1084124)</dd><br />
</dl><br />
<p><br />
We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed &beta;-Galactisidase assay. Reaction time was 2 hrs.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/f/f3/HokkaidoU_RBS_results1_800.png"><br />
<div><span class="bold">fig.1: Picture of &beta;-Galactosidase assay.</span> orange: strong LacZ&alpha; expression, yellow: weak expression.</div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/32/HokkaidoU2013_RBS_results2_new_800.png"><br />
<div><span class="bold">fig.2: LacZ&alpha; expression.</span> x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error. n=24.</div><br />
</div><br />
<br />
<p><br />
LacZ&alpha; protein hydrolyses substrate (Chlorophenol red-&beta;-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZ&alpha; expression is strong (fig.1).<br />
</p><br />
<p><br />
We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2).<br />
Construct with SD4 showed the strongest LacZ&alpha; activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs.<br />
</p><br />
<br />
<br />
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<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Conclusion"><div class="arrow-div"></div><span>Conclusion</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/File:HokkaidoU2013_RBS_results2_new_800.pngFile:HokkaidoU2013 RBS results2 new 800.png2013-10-28T16:05:52Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ResultsTeam:HokkaidoU Japan/RBS/Results2013-10-27T14:17:22Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Results</h1><br />
<p><br />
We made these constructs to measure translation efficiency of our RBSs. We assembled LacZ&alpha; to performed &beta;-Galactosidase assay.<br />
</p><br />
<dl><br />
<dt>positive control</dt><dd>pTet-B0034-LacZ&alpha;-dT</dd><br />
<dt>negative control</dt><dd>No insert</dd><br />
<dt>SD2</dt><dd>pTet-SD2-LacZ&alpha;-dT (BBa_K1084121)</dd><br />
<dt>SD4</dt><dd>pTet-SD4-LacZ&alpha;-dT (Bba_K1084122)</dd><br />
<dt>SD6</dt><dd>pTet-SD6-LacZ&alpha;-dT (Bba_K1084123)</dd><br />
<dt>SD8</dt><dd>pTet-SD8-LacZ&alpha;-dT (BBa_K1084124)</dd><br />
</dl><br />
<p><br />
We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed &beta;-Galactisidase assay. Reaction time was 2 hrs.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/f/f3/HokkaidoU_RBS_results1_800.png"><br />
<div><span class="bold">fig.1: Picture of &beta;-Galactosidase assay.</span> orange: strong LacZ&alpha; expression, yellow: weak expression.</div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png"><br />
<div><span class="bold">fig.2: LacZ&alpha; expression.</span> x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error. n=24.</div><br />
</div><br />
<br />
<p><br />
LacZ&alpha; protein hydrolyses substrate (Chlorophenol red-&beta;-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZ&alpha; expression is strong (fig.1).<br />
</p><br />
<p><br />
We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2).<br />
Construct with SD4 showed the strongest LacZ&alpha; activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs. Translation efficiency of SD4 and SD8 is the same as BBa_B0034, which is most used RBS.<br />
</p><br />
<br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Conclusion"><div class="arrow-div"></div><span>Conclusion</span></a><br />
</div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ResultsTeam:HokkaidoU Japan/RBS/Results2013-10-27T14:16:38Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<h1>Results</h1><br />
<p><br />
We made these constructs to measure translation efficiency of our RBSs. We assembled LacZ&alpha; to performed &beta;-Galactosidase assay.<br />
</p><br />
<dl><br />
<dt>positive control</dt><dd>pTet-B0034-LacZ&alpha;-dT</dd><br />
<dt>negative control</dt><dd>No insert</dd><br />
<dt>SD2</dt><dd>pTet-SD2-LacZ&alpha;-dT (BBa_K1084121)</dd><br />
<dt>SD4</dt><dd>pTet-SD4-LacZ&alpha;-dT (Bba_K1084122)</dd><br />
<dt>SD6</dt><dd>pTet-SD6-LacZ&alpha;-dT (Bba_K1084123)</dd><br />
<dt>SD8</dt><dd>pTet-SD8-LacZ&alpha;-dT (BBa_K1084124)</dd><br />
</dl><br />
<p><br />
We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed &beta;-Galactisidase assay. Reaction time was 2 hrs. We did this assay for 24 times.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/f/f3/HokkaidoU_RBS_results1_800.png"><br />
<div><span class="bold">fig.1: Picture of &beta;-Galactosidase assay.</span> orange: strong LacZ&alpha; expression, yellow: weak expression.</div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png"><br />
<div><span class="bold">fig.2: LacZ&alpha; expression.</span> x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error. n=24.</div><br />
</div><br />
<br />
<p><br />
LacZ&alpha; protein hydrolyses substrate (Chlorophenol red-&beta;-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZ&alpha; expression is strong (fig.1).<br />
</p><br />
<p><br />
We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2).<br />
Construct with SD4 showed the strongest LacZ&alpha; activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs. Translation efficiency of SD4 and SD8 is the same as BBa_B0034, which is most used RBS.<br />
</p><br />
<br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Conclusion"><div class="arrow-div"></div><span>Conclusion</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/SafetyTeam:HokkaidoU Japan/Safety2013-10-26T22:09:37Z<p>Barao: </p>
<hr />
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<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Safety</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
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<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>Basic Safety Questions for iGEM 2013</h1><br />
<div class="section"><br />
<h2>The chassis organism(s) we are using for this project.</h2><br />
<div class="answer"><br />
<ul><br />
<li><span class="italic">E.coli</span>(K 12) DH5&alpha;</li><br />
<li><span class="italic">E.coli</span>(K 12) JM109</li><br />
</ul><br />
</div><br />
<br />
<h3>Highest Risk Group Listed</h3><br />
<div class="answer"><br />
Risk Group 1<br />
</div><br />
<br />
<h2>This is a list of our new coding regions in our projects.</h2><br />
<div class="answer"><br />
<table><br />
<tr><br />
<th>Part number</th><br />
<th>Source of DNA</th><br />
<th>Species</th><br />
<th>Risk group</th><br />
<th>Function</th><br />
</tr><br />
<tr><td>BBa_K1084009</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084010</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084011</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084012</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084013</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084014</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084015</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084101</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084102</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084103</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084104</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
</table><br />
</div><br />
<br />
<br />
<h2>Description of the biological materials we are using in the lab.</h2><br />
<h3>Risks to the safety and health of team members or others working in the lab.</h3><br />
<div class="answer"><br />
Some materials pose risks to team members. For example, Ethidium Bromide is an intercalating agent so it must be used by with personal safety gear. All lab staff is trained according to safety manual provided by Hokkaido University, to prevent risks.<br />
We took on ourselves to compile a shortlist of often used dangerous materials and safety procedures in our project.<br />
</div><br />
<br />
<h3>Dangerous chemicals</h3><br />
<div class="answer"><br />
<dl><br />
<dt>Chloroform</dt><dd>corrosive and toxic : must be used in fume hood</dd><br />
<dt>Ethidium Bromide</dt><dd>intercalating agent : must be used with personal safety gear</dd><br />
<dt>Ethanol</dt><dd>flammable : must not be used near open flame or in large quantities</dd><br />
<dt>Liquid Nitrogen</dt><dd>cryogenic container and cryogenic gloves must be used</dd><br />
</dl><br />
</div><br />
<br />
<br />
<h3>Procedures and equipment</h3><br />
<div class="answer"><br />
<dl><br />
<dt>Agarose gel production</dt><dd>heating in sealed container (rupture risk), scalding hot and vicious during preparation (burn injury risk) - remove container lid before heating in microwave, use safety gear, wait for a few moments before removing from microwave</dd><br />
<dt>Benson burner</dt><dd>fire risk: DO NOT use flammable materials especially ethanol near open fire</dd><br />
<dt>Centrifuge</dt><dd>high velocity: balance appropriately, observe the machine till it reaches top velocity</dd><br />
<dt>Autoclave</dt><dd>high pressure: check the water level, DO NOT open when pressurized</dd><br />
<dt>UV radiation</dt><dd>damage to eyes and skin: use glove and UV box or UV shield</dd><br />
</dl><br />
</div><br />
<br />
<h3>Non-pathogenic bacteria (policy requires treating as pathogenic, as precaution)</h3><br />
<div class="answer"><br />
<ul><br />
<li>DH5&alpha;</li><br />
<li>JM109</li><br />
</ul><br />
<p><br />
Both of these are lab safe strains. As a precaution all materials coming in contact are sterilized before and after.<br />
Reference Federal Register, (1986) Vol. V1: 88, 6952–16985<br />
</p><br />
</div><br />
<br />
<h3>Safety equipment</h3><br />
<div class="answer"><br />
<ul><br />
<li>Gloves</li><br />
<li>Coats</li><br />
<li>Goggles</li><br />
<li>UV Box</li><br />
<li>UV shield</li><br />
</ul><br />
</div><br />
<br />
<h3>Waste disposal and sterilization</h3><br />
<div class="answer"><br />
<ul><br />
<li>All equipment and waste coming in contact with bacterial is sterilized by autoclave or bleach.</li><br />
<li>All chemicals compounds were disposed according to requirements for their disposal.</li><br />
<li>All table surface used for work were sterilized with 70% ethanol before and after a procedure.</li><br />
</ul><br />
</div><br />
<br />
<h3>Chemical Usage</h3><br />
<div class="answer"><br />
All chemical compounds were used according to their manuals and respective material safety data sheet<br />
</div><br />
<br />
<h2>Genetic material</h2><br />
<div class="answer"><br />
<p><br />
All genes used in this project come from non-pathogenic bacterial strains of <span class="italic">E.coli</span> or <span class="italic">R. eutropha</span>.<br />
Expressed proteins did not show any toxic effect to their host.<br />
Our biobricks do not have any foreseeable selective advantage if released to the environment.<br />
After consideration we could not find any usage pausing a security concern.<br />
</p><br />
</div><br />
<br />
<h3>Risks to the safety and health of the general public, if released by design or by accident.</h3><br />
<div class="answer"><br />
<p><br />
Some materials pose risks to the general public.<br />
For example, Ethanol is a flammable solution so it must not be used by open fire.<br />
Not to release those materials, all lab staff is trained according to safety manual provided by Hokkaido University.<br />
</p><br />
</div><br />
<br />
<h3>Risks to the environment, if released by design or by accident.</h3><br />
<div class="answer"><br />
<p><br />
The <span class="italic">E.coli</span> strains we use in our lab, are lab sage strains.<br />
As a precaution all materials coming in contact are sterilized before and after.<br />
Reference Federal Register, (1986) Vol. V1: 88, 6952-16985<br />
</p><br />
</div><br />
<br />
<h3>Risks to security through malicious misuse by individuals, groups, or countries.</h3><br />
<div class="answer"><br />
<p><br />
There is no foreseeable risk in misuse of our generated genetic material.<br />
Our generated genetic material performs basic functions in biology.<br />
However, it is impossible to guard against the incorporation of our parts in malicious settings.<br />
</p><br />
</div><br />
<br />
<h3>Risks which might arise when our project move from a small-scale lab study to become widely used as a commercial/industrial product.</h3><br />
<div class="answer"><br />
<p><br />
Our project is about optimizing the expression of genes. Our device does not contain a coding site.<br />
Therefore, risk will arise when other users assemble our parts with dangerous coding sites.<br />
We have to caution the user when assembling with dangerous coding sites.<br />
</p><br />
</div><br />
<br />
<h2>Design features to address safety risks.</h2><br />
<div class="answer"><br />
<p><br />
Our device only contains sequences that regulate the expression of genes.<br />
(Promoter, RBS, and terminator) Therefore, our device itself does not contain any safety risks and does not have design feature to address safety risks.<br />
</p><br />
</div><br />
<br />
<h2>Safety training we received.</h2><br />
<div class="answer"><br />
<p><br />
We all received a lecture class regarding gene recombination that were held in Hokkaido University.<br />
It is based on 'Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms'.<br />
</p><br />
</div><br />
<br />
<h2>Biosafety provisions</h2><br />
<h3>Link to our institution biosafety guidelines.</h3><br />
<div class="answer"><br />
<a href="http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html">http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html</a><br />
</div><br />
<br />
<h3>Our Institutional Biosafety Committee.</h3><br />
<div class="answer"><br />
<p><br />
We have a permission to engage in the experiments from the safety officer of genetic recombination of Hokkaido University.<br />
All members participating in the experiments are registered with this office.<br />
All members are trained according to the safety demands of safety officer of genetic recombination.<br />
</p><br />
</div><br />
<br />
<h3>Our country’s national biosafety regulations and guidelines</h3><br />
<div class="answer"><br />
<p><br />
Japan is participating in cartagena act. Please refer to a link below.<br><br />
<a href="http://www.bch.biodic.go.jp/english/cartagena/images/e_cartagena.pdf">http://www.bch.biodic.go.jp/english/cartagena/images/e_cartagena.pdf</a><br />
</p><br />
</div><br />
<br />
<h3>Biosafety Level rating of our lab.</h3><br />
<div class="answer"><br />
<p><br />
Our labs Bio safety level is 2.<br />
</p><br />
</div><br />
<br />
<h3>The Risk Group of our chassis organisms.</h3><br />
<div class="answer"><br />
<p><br />
The Risk Group of our chassis organisms is 1.<br />
</p><br />
</div><br />
<br />
<h2>Faculty Advisor</h2><br />
<div class="answer"><br />
Yamazaki Ken-ichi<br />
</div><br />
</div><br />
<br />
<br />
<!-- end contents / begin footer --><br />
</div><br />
</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/SafetyTeam:HokkaidoU Japan/Safety2013-10-26T22:07:52Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Safety</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>Basic Safety Questions for iGEM 2013</h1><br />
<div class="section"><br />
<h2>The chassis organism(s) we are using for this project.</h2><br />
<div class="answer"><br />
<ul><br />
<li><span class="italic">E.coli</span>(K 12) DH5&alpha;</li><br />
<li><span class="italic">E.coli</span>(K 12) JM109</li><br />
</ul><br />
</div><br />
<br />
<h3>Highest Risk Group Listed</h3><br />
<div class="answer"><br />
Risk Group 1<br />
</div><br />
<br />
<h2>This is a list of our new coding regions in our projects.</h2><br />
<div class="answer"><br />
<table><br />
<tr><br />
<th>Part number</th><br />
<th>Source of DNA</th><br />
<th>Species</th><br />
<th>Risk group</th><br />
<th>Function</th><br />
</tr><br />
<tr><td>BBa_K1084009</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084010</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084011</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084012</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084013</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084014</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084015</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084101</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084102</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084103</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084104</td><td>Synthesised, Sigma Alderich</td><td><span class="italic">E.coli</span></td><td>1</td><td>RBS</td></tr><br />
</table><br />
</div><br />
<br />
<br />
<h2>Description of the biological materials we are using in the lab.</h2><br />
<h3>Risks to the safety and health of team members or others working in the lab.</h3><br />
<div class="answer"><br />
Some materials pose risks to team members. For example, Ethidium Bromide is an intercalating agent so it must be used by with personal safety gear. All lab staff is trained according to safety manual provided by Hokkaido University, to prevent risks.<br />
We took on ourselves to compile a shortlist of often used dangerous materials and safety procedures in our project.<br />
</div><br />
<br />
<h3>Dangerous chemicals</h3><br />
<div class="answer"><br />
<dl><br />
<dt>Chloroform</dt><dd>corrosive and toxic : must be used in fume hood</dd><br />
<dt>Ethidium Bromide</dt><dd>intercalating agent : must be used with personal safety gear</dd><br />
<dt>Ethanol</dt><dd>flammable : must not be used near open flame or in large quantities</dd><br />
<dt>Liquid Nitrogen</dt><dd>cryogenic container and cryogenic gloves must be used</dd><br />
</dl><br />
</div><br />
<br />
<br />
<h3>Procedures and equipment</h3><br />
<div class="answer"><br />
<dl><br />
<dt>Agarose gel production</dt><dd>heating in sealed container (rupture risk), scalding hot and vicious during preparation (burn injury risk) - remove container lid before heating in microwave, use safety gear, wait for a few moments before removing from microwave</dd><br />
<dt>Benson burner</dt><dd>fire risk: DO NOT use flammable materials especially ethanol near open fire</dd><br />
<dt>Centrifuge</dt><dd>high velocity: balance appropriately, observe the machine till it reaches top velocity</dd><br />
<dt>Autoclave</dt><dd>high pressure: check the water level, DO NOT open when pressurized</dd><br />
<dt>UV radiation</dt><dd>damage to eyes and skin: use glove and UV box or UV shield</dd><br />
</dl><br />
</div><br />
<br />
<h3>Non-pathogenic bacteria (policy requires treating as pathogenic, as precaution)</h3><br />
<div class="answer"><br />
<ul><br />
<li>DH5&alpha;</li><br />
<li>JM109</li><br />
</ul><br />
<p><br />
Both of these are lab safe strains. As a precaution all materials coming in contact are sterilized before and after.<br />
Reference Federal Register, (1986) Vol. V1: 88, 6952–16985<br />
</p><br />
</div><br />
<br />
<h3>Safety equipment</h3><br />
<div class="answer"><br />
<ul><br />
<li>Gloves</li><br />
<li>Coats</li><br />
<li>Goggles</li><br />
<li>UV Box</li><br />
<li>UV shield</li><br />
</ul><br />
</div><br />
<br />
<h3>Waste disposal and sterilization</h3><br />
<div class="answer"><br />
<ul><br />
<li>All equipment and waste coming in contact with bacterial is sterilized by autoclave or bleach.</li><br />
<li>All chemicals compounds were disposed according to requirements for their disposal.</li><br />
<li>All table surface used for work were sterilized with 70% ethanol before and after a procedure.</li><br />
</ul><br />
</div><br />
<br />
<h3>Chemical Usage</h3><br />
<div class="answer"><br />
All chemical compounds were used according to their manuals and respective material safety data sheet<br />
</div><br />
<br />
<h2>Genetic material</h2><br />
<div class="answer"><br />
<p><br />
All genes used in this project come from non-pathogenic bacterial strains of E. coli or R. eutropha.<br />
Expressed proteins did not show any toxic effect to their host.<br />
Our biobricks do not have any foreseeable selective advantage if released to the environment.<br />
After consideration we could not find any usage pausing a security concern.<br />
</p><br />
</div><br />
<br />
<h3>Risks to the safety and health of the general public, if released by design or by accident.</h3><br />
<div class="answer"><br />
<p><br />
Some materials pose risks to the general public.<br />
For example, Ethanol is a flammable solution so it must not be used by open fire.<br />
Not to release those materials, all lab staff is trained according to safety manual provided by Hokkaido University.<br />
</p><br />
</div><br />
<br />
<h3>Risks to the environment, if released by design or by accident.</h3><br />
<div class="answer"><br />
<p><br />
The E. coli strains we use in our lab, are lab sage strains.<br />
As a precaution all materials coming in contact are sterilized before and after.<br />
Reference Federal Register, (1986) Vol. V1: 88, 6952-16985<br />
</p><br />
</div><br />
<br />
<h3>Risks to security through malicious misuse by individuals, groups, or countries.</h3><br />
<div class="answer"><br />
<p><br />
There is no foreseeable risk in misuse of our generated genetic material.<br />
Our generated genetic material performs basic functions in biology.<br />
However, it is impossible to guard against the incorporation of our parts in malicious settings.<br />
</p><br />
</div><br />
<br />
<h3>Risks which might arise when our project move from a small-scale lab study to become widely used as a commercial/industrial product.</h3><br />
<div class="answer"><br />
<p><br />
Our project is about optimizing the expression of genes. Our device does not contain a coding site.<br />
Therefore, risk will arise when other users assemble our parts with dangerous coding sites.<br />
We have to caution the user when assembling with dangerous coding sites.<br />
</p><br />
</div><br />
<br />
<h2>Design features to address safety risks.</h2><br />
<div class="answer"><br />
<p><br />
Our device only contains sequences that regulate the expression of genes.<br />
(Promoter, RBS, and terminator) Therefore, our device itself does not contain any safety risks and does not have design feature to address safety risks.<br />
</p><br />
</div><br />
<br />
<h2>Safety training we received.</h2><br />
<div class="answer"><br />
<p><br />
We all received a lecture class regarding gene recombination that were held in Hokkaido University.<br />
It is based on 'Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms'.<br />
</p><br />
</div><br />
<br />
<h2>Biosafety provisions</h2><br />
<h3>Link to our institution biosafety guidelines.</h3><br />
<div class="answer"><br />
<a href="http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html">http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html</a><br />
</div><br />
<br />
<h3>Our Institutional Biosafety Committee.</h3><br />
<div class="answer"><br />
<p><br />
We have a permission to engage in the experiments from the safety officer of genetic recombination of Hokkaido University.<br />
All members participating in the experiments are registered with this office.<br />
All members are trained according to the safety demands of safety officer of genetic recombination.<br />
</p><br />
</div><br />
<br />
<h3>Our country’s national biosafety regulations and guidelines</h3><br />
<div class="answer"><br />
<p><br />
Japan is participating in cartagena act. Please refer to a link below.<br><br />
<a href="http://www.bch.biodic.go.jp/english/cartagena/images/e_cartagena.pdf">http://www.bch.biodic.go.jp/english/cartagena/images/e_cartagena.pdf</a><br />
</p><br />
</div><br />
<br />
<h3>Biosafety Level rating of our lab.</h3><br />
<div class="answer"><br />
<p><br />
Our labs Bio safety level is 2.<br />
</p><br />
</div><br />
<br />
<h3>The Risk Group of our chassis organisms.</h3><br />
<div class="answer"><br />
<p><br />
The Risk Group of our chassis organisms is 1.<br />
</p><br />
</div><br />
<br />
<h2>Faculty Advisor</h2><br />
<div class="answer"><br />
Yamazaki Ken-ichi<br />
</div><br />
</div><br />
<br />
<br />
<!-- end contents / begin footer --><br />
</div><br />
</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/SafetyTeam:HokkaidoU Japan/Safety2013-10-26T22:06:39Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Safety</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h1>Basic Safety Questions for iGEM 2013</h1><br />
<div class="section"><br />
<h2>The chassis organism(s) we are using for this project.</h2><br />
<div class="answer"><br />
<ul><br />
<li><span class="italic">E.coli</span>(K 12) DH5&alpha;</li><br />
<li><span class="italic">E.coli</span>(K 12) JM109</li><br />
</ul><br />
</div><br />
<br />
<h3>Highest Risk Group Listed</h3><br />
<div class="answer"><br />
Risk Group 1<br />
</div><br />
<br />
<h2>This is a list of our new coding regions in our projects.</h2><br />
<div class="answer"><br />
<table><br />
<tr><br />
<th>Part number</th><br />
<th>Source of DNA</th><br />
<th>Species</th><br />
<th>Risk group</th><br />
<th>Function</th><br />
</tr><br />
<tr><td>BBa_K1084009</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084010</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084011</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084012</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084013</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084014</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084015</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>Promoter</td></tr><br />
<tr><td>BBa_K1084101</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084102</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084103</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>RBS</td></tr><br />
<tr><td>BBa_K1084104</td><td>Synthesised, Sigma Alderich</td><td>E.coli</td><td>1</td><td>RBS</td></tr><br />
</table><br />
</div><br />
<br />
<br />
<h2>Description of the biological materials we are using in the lab.</h2><br />
<h3>Risks to the safety and health of team members or others working in the lab.</h3><br />
<div class="answer"><br />
Some materials pose risks to team members. For example, Ethidium Bromide is an intercalating agent so it must be used by with personal safety gear. All lab staff is trained according to safety manual provided by Hokkaido University, to prevent risks.<br />
We took on ourselves to compile a shortlist of often used dangerous materials and safety procedures in our project.<br />
</div><br />
<br />
<h3>Dangerous chemicals</h3><br />
<div class="answer"><br />
<dl><br />
<dt>Chloroform</dt><dd>corrosive and toxic : must be used in fume hood</dd><br />
<dt>Ethidium Bromide</dt><dd>intercalating agent : must be used with personal safety gear</dd><br />
<dt>Ethanol</dt><dd>flammable : must not be used near open flame or in large quantities</dd><br />
<dt>Liquid Nitrogen</dt><dd>cryogenic container and cryogenic gloves must be used</dd><br />
</dl><br />
</div><br />
<br />
<br />
<h3>Procedures and equipment</h3><br />
<div class="answer"><br />
<dl><br />
<dt>Agarose gel production</dt><dd>heating in sealed container (rupture risk), scalding hot and vicious during preparation (burn injury risk) - remove container lid before heating in microwave, use safety gear, wait for a few moments before removing from microwave</dd><br />
<dt>Benson burner</dt><dd>fire risk: DO NOT use flammable materials especially ethanol near open fire</dd><br />
<dt>Centrifuge</dt><dd>high velocity: balance appropriately, observe the machine till it reaches top velocity</dd><br />
<dt>Autoclave</dt><dd>high pressure: check the water level, DO NOT open when pressurized</dd><br />
<dt>UV radiation</dt><dd>damage to eyes and skin: use glove and UV box or UV shield</dd><br />
</dl><br />
</div><br />
<br />
<h3>Non-pathogenic bacteria (policy requires treating as pathogenic, as precaution)</h3><br />
<div class="answer"><br />
<ul><br />
<li>DH5&alpha;</li><br />
<li>JM109</li><br />
</ul><br />
<p><br />
Both of these are lab safe strains. As a precaution all materials coming in contact are sterilized before and after.<br />
Reference Federal Register, (1986) Vol. V1: 88, 6952–16985<br />
</p><br />
</div><br />
<br />
<h3>Safety equipment</h3><br />
<div class="answer"><br />
<ul><br />
<li>Gloves</li><br />
<li>Coats</li><br />
<li>Goggles</li><br />
<li>UV Box</li><br />
<li>UV shield</li><br />
</ul><br />
</div><br />
<br />
<h3>Waste disposal and sterilization</h3><br />
<div class="answer"><br />
<ul><br />
<li>All equipment and waste coming in contact with bacterial is sterilized by autoclave or bleach.</li><br />
<li>All chemicals compounds were disposed according to requirements for their disposal.</li><br />
<li>All table surface used for work were sterilized with 70% ethanol before and after a procedure.</li><br />
</ul><br />
</div><br />
<br />
<h3>Chemical Usage</h3><br />
<div class="answer"><br />
All chemical compounds were used according to their manuals and respective material safety data sheet<br />
</div><br />
<br />
<h2>Genetic material</h2><br />
<div class="answer"><br />
<p><br />
All genes used in this project come from non-pathogenic bacterial strains of E. coli or R. eutropha.<br />
Expressed proteins did not show any toxic effect to their host.<br />
Our biobricks do not have any foreseeable selective advantage if released to the environment.<br />
After consideration we could not find any usage pausing a security concern.<br />
</p><br />
</div><br />
<br />
<h3>Risks to the safety and health of the general public, if released by design or by accident.</h3><br />
<div class="answer"><br />
<p><br />
Some materials pose risks to the general public.<br />
For example, Ethanol is a flammable solution so it must not be used by open fire.<br />
Not to release those materials, all lab staff is trained according to safety manual provided by Hokkaido University.<br />
</p><br />
</div><br />
<br />
<h3>Risks to the environment, if released by design or by accident.</h3><br />
<div class="answer"><br />
<p><br />
The E. coli strains we use in our lab, are lab sage strains.<br />
As a precaution all materials coming in contact are sterilized before and after.<br />
Reference Federal Register, (1986) Vol. V1: 88, 6952-16985<br />
</p><br />
</div><br />
<br />
<h3>Risks to security through malicious misuse by individuals, groups, or countries.</h3><br />
<div class="answer"><br />
<p><br />
There is no foreseeable risk in misuse of our generated genetic material.<br />
Our generated genetic material performs basic functions in biology.<br />
However, it is impossible to guard against the incorporation of our parts in malicious settings.<br />
</p><br />
</div><br />
<br />
<h3>Risks which might arise when our project move from a small-scale lab study to become widely used as a commercial/industrial product.</h3><br />
<div class="answer"><br />
<p><br />
Our project is about optimizing the expression of genes. Our device does not contain a coding site.<br />
Therefore, risk will arise when other users assemble our parts with dangerous coding sites.<br />
We have to caution the user when assembling with dangerous coding sites.<br />
</p><br />
</div><br />
<br />
<h2>Design features to address safety risks.</h2><br />
<div class="answer"><br />
<p><br />
Our device only contains sequences that regulate the expression of genes.<br />
(Promoter, RBS, and terminator) Therefore, our device itself does not contain any safety risks and does not have design feature to address safety risks.<br />
</p><br />
</div><br />
<br />
<h2>Safety training we received.</h2><br />
<div class="answer"><br />
<p><br />
We all received a lecture class regarding gene recombination that were held in Hokkaido University.<br />
It is based on 'Act on the Conservation and Sustainable Use of Biological Diversity through Regulations on the Use of Living Modified Organisms'.<br />
</p><br />
</div><br />
<br />
<h2>Biosafety provisions</h2><br />
<h3>Link to our institution biosafety guidelines.</h3><br />
<div class="answer"><br />
<a href="http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html">http://www.hokudai.ac.jp/jimuk/reiki/reiki_honbun/u010RG00000583.html</a><br />
</div><br />
<br />
<h3>Our Institutional Biosafety Committee.</h3><br />
<div class="answer"><br />
<p><br />
We have a permission to engage in the experiments from the safety officer of genetic recombination of Hokkaido University.<br />
All members participating in the experiments are registered with this office.<br />
All members are trained according to the safety demands of safety officer of genetic recombination.<br />
</p><br />
</div><br />
<br />
<h3>Our country’s national biosafety regulations and guidelines</h3><br />
<div class="answer"><br />
<p><br />
Japan is participating in cartagena act. Please refer to a link below.<br><br />
<a href="http://www.bch.biodic.go.jp/english/cartagena/images/e_cartagena.pdf">http://www.bch.biodic.go.jp/english/cartagena/images/e_cartagena.pdf</a><br />
</p><br />
</div><br />
<br />
<h3>Biosafety Level rating of our lab.</h3><br />
<div class="answer"><br />
<p><br />
Our labs Bio safety level is 2.<br />
</p><br />
</div><br />
<br />
<h3>The Risk Group of our chassis organisms.</h3><br />
<div class="answer"><br />
<p><br />
The Risk Group of our chassis organisms is 1.<br />
</p><br />
</div><br />
<br />
<h2>Faculty Advisor</h2><br />
<div class="answer"><br />
Yamazaki Ken-ichi<br />
</div><br />
</div><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Shuffling_Kit/How_To_UseTeam:HokkaidoU Japan/Shuffling Kit/How To Use2013-10-26T22:03:19Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
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<div id="common-header-bottom-background"><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Optimization Kit</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
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<br />
<br />
<h1>How to use</h1><br />
<h2>What users should prepare</h2><br />
<p><br />
To use the kit, the protein sequence you chose must have specific prefix/suffix which contains a BsaI site and produce overhang. Therefore, users must design a primer to add it. To reduce time and trouble, we automated the design by creating program <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">"Primer Designer for Maestro"!</a><br />
</p><br />
<br />
<h2>Promoter Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our Promoter Selector consists of 5 different plasmids (table.1). Each has a promoter with a different strength. Downstream of the RBS there is a BsaI site to insert the protein sequence. There is a color expression construct downstream of protein insertion site (fig.1). Each color is paired with different strength promoter. The pairings are shown on the table below.<br />
</p><br />
<div class="fig fig800"><br />
<style type="text/css"><br />
table { border: 1px solid #a4a4a4; margin: 0 auto; }<br />
td, th { text-align: center; }<br />
</style><br />
<table><br />
<tr><br />
<th>Part number</th><th>Promoter</th><th>Promoter strength</th><th>Paired protein</th><th>Protein color</th><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084501">BBa_K1084501</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084001">BBa_K1084001</a></td><td>Strongest</td><td>amilGFP</td><td>yellowish green</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084502">BBa_K1084502</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084002">BBa_K1084002</a></td><td>Stronger</td><td>aeBlue</td><td>strong blue</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084503">BBa_K1084503</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084005">BBa_K1084005</a></td><td>Medium</td><td>amilCP</td><td>Purple</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084504">BBa_K1084504</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084009">BBa_K1084009</a></td><td>Weaker</td><td>mRFP</td><td>Pink</td><br />
</tr><br />
<tr><br />
<td><a href="http://parts.igem.org/Part:BBa_K1084505">BBa_K1084505</a></td><td><a href="http://parts.igem.org/Part:BBa_K1084010">BBa_K1084010</a></td><td>Weakest</td><td>eforRED</td><td>red</td><br />
</tr><br />
</table><br />
<br />
<div>table.1</div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/95/HokkaidoU_2013_Fig5_new_800.png"><br />
<div>fig.1</div><br />
</div><br />
<p><br />
The color expression is induced by IPTG. If you don't need the color expressing construct, you can remove it by using PstI.<br />
LacZ&alpha; reporter is placed between two BsaI sites (fig.2). The LacZ&alpha; expressing construct will be replaced by chosen sequence using BsaI.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cd/HokkaidoU_opti_fig6_2.png"><br />
<div>fig.2</div><br />
</div><br />
<br />
<h2>How it works</h2><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/2/27/HokkaidoU2013_optimization_Fig7_ver2_400.png"><br />
<div>fig.3</div><br />
</div><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence (fig.3). PCR with primers designed with <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">our program</a> should do the trick.<br />
</p><br />
<br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all our Promoter Selector together (fig.4). This is accomplished by "Golden Gate Assembly" reaction. The detailed recipe is shown in Engler (2009)<sup><a href="#cite-1">[1]</a></sup>. All the protein coding sequence will be inserted in the plasmid.<br />
<!-- add more detail --><br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a5/HokkaidoU2013_optimization_fig8slecat.png"><br />
<div>fig.4</div><br />
</div><br />
<br />
<p><br />
3. You should get the construct shown below (fig.5).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/6/66/HokkaidoU_2013_Fig9_new_800.png"><br />
<div>fig.5</div><br />
</div><br />
<p><br />
4. Transform the ligated DNA to <span class="italic">E. coli</span>, and spread it on plate containing IPTG. Then, you will get colonies with five colors (fig.6). The colors are paired with the promoters, so you will know which promoter is used instantly!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/8/88/HokkaidoU2013_optimization_Fig10_new_800.png"><br />
<div>fig.6</div><br />
</div><br />
<p><br />
5. Pick up the colonies and add to culture. Assay to check the production of protein. When you don't want the colors to be expressed, you can remove the color expressing construct by chosen sequence using PstI (fig.7).<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/f/fb/HokkaidoU_2013_Fig11_new_800.png"><br />
<div>fig.7</div><br />
</div><br />
<br />
<br />
<h2>RBS Selector</h2><br />
<h3>What our kit contains</h3><br />
<p><br />
Our kit contains tandem RBS (fig.8) and acceptor plasmid (fig.9).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/22/RBS_strength_2_HokkaidoU_2013.png"><br />
<div>fig.8</div><br />
</div><br />
<p><br />
In this part, 4 strength levels of RBSs[BBa_K1084101, BBa_ K1084102, BBa_ K1084103, BBa_ K1084104] are connected in tandem. To optimize up to 3 coding sequence expressions in the operon this part has three sets of RBSs with different overhangs are connected together.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU2013_optimization_Fig13_800.png"><br />
<div>fig.9 This is the acceptor part of the RBS and protein coding region. It has a BsaI site for the parts to be assembled.</div><br />
</div><br />
</p><br />
<br />
<h3>How to use</h3><br />
<br />
<p><br />
1. Have BsaI site and specific overhang added to your protein sequence. Again, our program should help your primers design (fig.10). Also when you want to optimize more than one protein coding sites, add BsaI sites and overhang to them too. Be careful not to choose the same overhangs.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/HokkaidoU2013_optimization_Fig14_800.png"><br />
<div>fig.10</div><br />
</div><br />
<p><br />
2.<br />
Digest and ligate your protein coding sequence and all RBS Selector together. This reaction is also accomplished by Golden Gate Assembly. DNA fragments will be assembled in the desired order (fig.11).<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/7/7d/HokkaidoU2013_optimization_Fig15_ver.2_800.png"><br />
<div>fig.11</div><br />
</div><br />
<br />
<p><br />
3. Transform the ligated DNA to <span class="italic">E. coli</span>. If you are optimizing three different proteins, you will get 64 different kinds of constructs.<br />
</p><br />
<br />
<p><br />
We will submit these standard methods as RFC to BioBrick Foundation.<br />
</p><br />
<br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">C. Engler <span class="italic">et al.</span> Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009) PLoS ONE</li><br />
</ol><br />
<br />
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<a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer"><div class="arrow-div"></div><span>Primer Designer</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/Overview/BackgroundTeam:HokkaidoU Japan/Overview/Background2013-10-26T21:59:37Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">Overview</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
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<div id="hokkaidou-contents"><br />
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<h1>Background</h1><br />
<br />
<p><br />
Synthetic biology is one of the most interesting fields in 21st century.<br />
Its goal is to comprehend and reproduce the marvels of living things.<br />
As members of syn-bio community, a lot of iGEMers have found interesting proteins.<br />
However, the focuses of these projects are “qualities” of proteins.<br />
As well as the uniqueness of proteins, the expression levels of proteins contribute to the marvelous functions of living things.<br />
The next goal of syn-bio is to control “quantities” of proteins.<br />
</p><br />
<br />
<p><br />
To control protein quantities in the organism, we must control transcription, a step from DNA to mRNA and translation, a step from mRNA to protein.<br />
Transcription is regulated by promoter region.<br />
Translation is regulated by ribosome binding site: RBS.<br />
Depending on promoters, the transcription rate varies about 1000 fold.<br />
Similarly, depending on RBSs, the translation rate about 100 fold.<br />
So, to control quantity, it is essential to figure out the characteristics of these expression-regulatory regions and adjust them.<br />
</p><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8a/HokkaidoU2013_Motivation_figure4_new.png"><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/28/HokkaidoU2013_Motivation_figure5.png"><br />
</div><br />
<p><br />
Recently, many studies have been done to theoretically predict the amount of gene expression by the sequences of expression-regulatory regions.<br />
There are some accurate in vivo expression efficiency predictions.<br />
Besides, even if you were able to predict it exactly, you could not adjust it to an optimal level because you don't know the best rate for the bacteria.<br />
</p><br />
<br />
<p><br />
Suppose you are planning to express beneficial product, like antibodies.<br />
You may think that the strongest promoter and RBS lead the maximal yield of the proteins.<br />
But actually, selecting the strongest one isn't always the best strategy.<br />
Overexpressed proteins can be perceived as detrimental by bacterial immune system and packed into inclusion bodies.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_Motivation_figure1.png"><br />
</div><br />
<p><br />
Another example is a multiple expression, such as mixing color of chromoprotein.<br />
Theoretically, three different chromoproteins can express any kind of colors by fixing each expression level.<br />
But this adjustment is too sensitive to be made instinctively.<br />
</p><br />
<div class="fig fig300"><br />
<img src="https://static.igem.org/mediawiki/2013/7/76/HokkaidoU2013_Motivation_figure3_big.png"><br />
</div><br />
<p><br />
Over these challenging problems in syn-bio, we put forward one solution like a Columbus's egg.<br />
"No more prediction, let's experiment!" ---- that is, we should try several promoters or RBSs and select the best one or the best combination.<br />
We don't know the best answer, but bacteria do.<br />
</p><br />
<br />
<br />
<p><br />
This is surely the best way, but trying every pattern costs a lot of time and labor.<br />
We overcame this obstacle by using a one-pot DNA shuffling method, namely, "Golden Gate Assembly".<br />
And then, we made two kits to optimize transcription and translation.<br />
For this kit, we made artificial promoter and RBS families.<br />
They are created based on concrete philosophies and characterized well.<br />
Please refer <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Promoter">Promoter</a> and <a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS">RBS</a> for details.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/HokkaidoU2013_Motivation_figure2.png"><br />
</div><br />
<p><br />
We named these kits 'Promoter Selector' and 'RBS selector'.<br />
<dl><br />
<dt>Promoter Selector</dt><dd>Promoter Selector is for adjustment of relatively simple expression, like a production of antibodies reffered above.</dd><br />
<dt>RBS Selector</dt><dd>RBS Selector is for adjustment of multiple expressions, like complex metabolisms.</dd><br />
</dl><br />
We named these two devices ''Maestro <span class="italic">E.coli</span>'' Random Operon Shuffling Kit! As a maestro harmonizes music, let's harmonize proteins in the bacteria! <br />
<br />
</p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-22T11:55:05Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png"><br />
</div><br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla <span class="italic">et al.</span> (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for regulating expression.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, <span class="italic">et al.</span> Coding-Sequence Determinants of Gene Expression in <span class="italic">Escherichia coli</span> (2009) Science</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ResultsTeam:HokkaidoU Japan/RBS/Results2013-10-22T11:52:54Z<p>Barao: </p>
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<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
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<!-- end header / begin contents --><br />
<h1>Results</h1><br />
<p><br />
We made these constructs to measure translation efficiency of our RBSs. We assembled LacZ&alpha; to performed &beta;-Galactosidase assay.<br />
</p><br />
<dl><br />
<dt>positive control</dt><dd>pTET-B0034-LacZ&alpha;-dT</dd><br />
<dt>negative control</dt><dd>No insert</dd><br />
<dt>SD2</dt><dd>pTET-SD2-LacZ&alpha;-dT (BBa_K1084121)</dd><br />
<dt>SD4</dt><dd>pTET-SD4-LacZ&alpha;-dT (Bba_K1084122)</dd><br />
<dt>SD6</dt><dd>pTET-SD6-LacZ&alpha;-dT (Bba_K1084123)</dd><br />
<dt>SD8</dt><dd>pTET-SD8-LacZ&alpha;-dT (BBa_K1084124)</dd><br />
</dl><br />
<p><br />
We moved our constructs to pSB3K3 plasmid, cultured for 9 hrs in 5 mL LB media round tubes, and performed &beta;-Galactisidase assay. Reaction time was 2 hrs. We did this assay for 24 times.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/f/f3/HokkaidoU_RBS_results1_800.png"><br />
<div>fig.1: Picture of &beta;-Galactosidase assay. orange: strong LacZ&alpha; expression, yellow: weak expression.</div><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png"><br />
<div>fig.2: LacZ&alpha; expression. x axis: sample name, y axis: standardized expression (positive control = 1.0), bar: standard error.</div><br />
</div><br />
<br />
<p><br />
LacZ&alpha; protein hydrolyses substrate (Chlorophenol red-&beta;-D-galactopyranoside), which is yellow. Product of this reaction has red color. Therefore, solution will turn deep orange if LacZ&alpha; expression is strong (fig.1).<br />
</p><br />
<p><br />
We measured absorbance of catalytic reaction solution at 595 nm, standardized using positive control and made into graph (fig.2).<br />
Construct with SD4 showed the strongest LacZ&alpha; activity. Second strongest was the SD8, followed by SD6. SD2 had the weakest activity. There is a significant difference in translation efficiency of our RBSs. Translation efficiency of SD4 and SD8 is the same as BBa_B0034, which is most used RBS.<br />
</p><br />
<br />
<br />
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<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
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<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Conclusion"><div class="arrow-div"></div><span>Conclusion</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/MethodsTeam:HokkaidoU Japan/RBS/Methods2013-10-22T11:51:26Z<p>Barao: </p>
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<h1 id="common-header-title">Maestro E.coli</h1><br />
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<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h2>RBS family parts</h2><br />
<p><br />
We constructed new RBS family, SD2, SD4, SD6, SD8.<br />
These RBSs have Enhancer sequence (GCTCTTTAACAATTTATCA) and SD sequence (SD2:GG, SD4:GAGG, SD6:AGGAGG, SD8:TAAGGAGG).<br />
We constructed SD8 from synthetic oligos (forward:SD8-f, reverse:SD8-r).<br />
We constructed SD2, SD4, SD6 by PCR (forward:EX-f, reverse:SD2-r, SD4-r, SD6-r, template:SD8).<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ab/HokkaidoU_RBS_methods1_800.png"><br />
<div>fig.1: oligos; RED: enhancer sequence, BLUE: SD sequence.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/9/9e/HokkaidoU_RBS_methods2_400.png"><br />
<div>fig.2: RBS construction</div><br />
</div><br />
<br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1a/HokkaidoU2013_RBS_methods3revision_400.png"><br />
<div>fig.3: our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<h2>Assay</h2><br />
<p><br />
We ligated TetR repressible promoter (pTET), each of the new RBSs', LacZ&alpha; and double terminator.<br />
Using this construct we performed &beta;-Galactosidase assay.<br />
</p><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T11:50:30Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, <span class="italic">et al.</span> Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, <span class="italic">et al.</span> Translation initiation region sequence preferences in <span class="italic">Escherichia coli</span> (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T11:42:24Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in <span class="italic">Escherichia coli</span> (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-22T11:16:51Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png"><br />
</div><br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla et al (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for regulating expression.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, et al, Coding-Sequence Determinants of Gene Expression in <span class="italic">Escherichia coli</span> (2009) Science</li><br />
</ol><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Results"><div class="arrow-div"></div><span>Results</span></a><br />
</div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/ConclusionTeam:HokkaidoU Japan/RBS/Conclusion2013-10-22T11:12:50Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<h2>Conclusion</h2><br />
<p><br />
We were successful at making four RBS set with four strength levels.<br />
Construct with SD4 showed the strongest &beta;-Galactosidase activity.<br />
Second strongest was the SD8, followed by B0034 and SD6. SD2 had the weakest activity.<br />
</p><br />
<p><br />
Though we synthesized the RBSs based on previous reports, we got unexpected results.<br />
Preciously SD6 was reported as the strongest. However our results indicated SD4.<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1d/HokkaidoU2013_RBS_Conclusion_800.png"><br />
</div><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3d/HokkaidoU_RBS_results2_800.png"><br />
</div><br />
<p><br />
The sequence we synthesized was completely the same.<br />
Both plasmids had low copy number.<br />
One thing we changed was the reporter gene.<br />
In the previous experiments, GFP was used for the reporter.<br />
In contrast, we used LacZ&alpha;.<br />
The expected strength differed only by changing the coding sequence.<br />
</p><br />
<p><br />
It is well known fact that mRNA makes a secondary structure. The secondary structure of RNA takes an important role in the process of life.<br />
Identically, the secondary structure and the folding of mRNA is an important factor in translation efficiency.<br />
Kudla et al (2009) showed that translation efficiency is determined by factors in Coding-Sequence.<br />
Overall, the translational efficiency is a correlation between RBS sequence and the Coding-Sequence.<br />
</p><br />
<p><br />
Our results show different levels of translation efficiency.<br />
We should try and repeat our assays with other reporter genes.<br />
The translation efficiency might change by choosing different coding sequences.<br />
The translation efficiency not depended on RBS but also influenced by coding sequence.<br />
This fact complicates designing biological devices.<br />
</p><br />
<p><br />
When regulating the expression it is important to have variability in RBSs strength.<br />
Our set RBSs proved to have different efficiencies in both GFP and LacZ&alpha; assays.<br />
From our results and the previous research we can expect that our RBSs will show variability in translational efficiency using any coding site.<br />
Therefore, we made a useful set of RBSs for expression optimization.<br />
</p><br />
<ol class="citation-list"><br />
<li id="cite-1">Grzegorz Kudla, et al, Coding-Sequence Determinants of Gene Expression in Escherichia coli (2009) Science</li><br />
</ol><br />
<br />
<br />
<br />
<div id="prev-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Results"><div class="arrow-div"></div><span>Results</span></a><br />
</div><br />
<br />
<br />
<!-- end contents / begin footer --><br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T10:47:32Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in <i>Escherichia coli</i> (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
<!-- end contents / begin footer --><br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T10:46:41Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in <b>Escherichia coli</b> (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T10:45:37Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
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<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in <i> Escherichia coli </i> (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T10:44:43Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in <i>Escherichia coli</i> (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
<!-- end contents / begin footer --><br />
</div><br />
</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T10:39:40Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector, we constructed well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in Escherichia coli (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
<!-- end contents / begin footer --><br />
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</div><br />
</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBSTeam:HokkaidoU Japan/RBS2013-10-22T10:38:38Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<p><br />
To make RBS selector a better one, we made well-selected sets of RBSs.<br />
For parts controlling gene expression such as promoters or RBSs, it is desired that their prospective functions are explainable.<br />
We wanted our parts to have, "transparent structure", "reliable function" and "reproducibility".<br />
Thus, when making our new parts, we decided to change only one region in mRNA.<br />
</p><br />
<p><br />
Ribosome binding site (RBS) is located upstream of initiation codon in mRNA. Translation efficiency depends on RBS sequence.<br />
RBS has Shine-Dalgarno sequence (SD).<br />
SD binds Anti-Shine-Dalgarno sequence (ASD) on ribosomal 30S subunit.<br />
Then initiation codon binds with fMet-tRNA anticodon and the translation will begin.<br />
SD-ASD binding strength is important for translation efficiency.<br />
However, there are results that show RBS binding to 30S subunit even if there is no SD sequence.<br />
</p><br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/HokkaidoU_RBS_background1_400.png"><br />
<div>fig.1: Ribosome and mRNA. First, S1 protein binds A/U rich sequence. Then, ASD binds SD.</div><br />
</div><br />
<p><br />
There is another place that binds with the ribosome in mRNA.<br />
Upstream of the SD sequence, there is an A/U rich sequence.<br />
This A/U rich sequence binds with S1 protein, which is one of proteins that makes 30S ribosome<sup><a href="#cite-1">[1]</a></sup>.<br />
The sequence has an important role to make the translation initiation complex.<br />
To make it, mRNA first has to bind with 30S ribosome which results to the binding of SD and ASD.<br />
Then A/U rich sequence and S1 protein binds together.<br />
The loose binding with A/U rich sequence and S1 protein, leads binding with SD and ASD (fig.1).<br />
Thus, this A/U rich sequence is called translational "enhancer"!<br />
</p><br />
<p><br />
Vimberg<sup><a href="#cite-2">[2]</a></sup> constructed RBSs by changing enhancer sequence and SD length (fig.2).<br />
Although SD length was changed, there wasn't big difference in translation efficiency among RBSs' without enhancer (fig.3).<br />
But big difference appeared in enhancer RBSs when SD length was changed (fig.4).<br />
Strong SDs are stimulated and weak SDs are repressed by A/U rich enhancer.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/9/92/HokkaidoU_RBS_background2_800.png"><br />
<div>fig.2: Various enhancer and SD. Vimberg combined 3 enhancers and 10 SDs and measured GFP expression.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c2/HokkaidoU2013_RBS_Background3.png"><br />
<div>fig.3: GFP expression in No enhancer RBSs.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/2/2d/HokkaidoU2013_RBS_Background4.png"><br />
<div>fig.4: GFP expression in A/U rich RBSs.</div><br />
</div><br />
<br />
<p><br />
We decided to constructed 4 new RBSs based on Vimberg.<br />
These RBSs have A/U rich enhancer.<br />
To change the translation efficiencies we varied the length of SD sequence.<br />
</p><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">B. S. Laursen, H. P. Sørensen, et al. Initiation of Protein Synthesis in Bacteria (2005) Microbiol. Mol. Biol. Rev.</li><br />
<li id="cite-2">V. Vimberg, A. Tats, et al. Translation initiation region sequence preferences in Escherichia coli (2007) BMC Molecular Biology</li><br />
</ol><br />
<br />
<br />
<div id="next-page"><br />
<a href="https://2013.igem.org/Team:HokkaidoU_Japan/RBS/Methods"><div class="arrow-div"></div><span>Methods</span></a><br />
</div><br />
<br />
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</html><br />
{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/Team:HokkaidoU_Japan/RBS/MethodsTeam:HokkaidoU Japan/RBS/Methods2013-10-20T14:53:32Z<p>Barao: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header_Maestro}}<br />
<html><br />
<div id="common-header-bottom-background"><br />
<div class="wrapper"><br />
<h1 id="common-header-title">Maestro E.coli</h1><br />
<h2 id="common-header-subtitle">RBS</h2><br />
<img id="common-header-img" src="https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png"><br />
</div><br />
</div><br />
<br />
<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<!-- end header / begin contents --><br />
<br />
<br />
<h2>RBS family parts</h2><br />
<p><br />
We constructed new RBS family, SD2, SD4, SD6, SD8.<br />
These RBSs have Enhancer sequence (GCTCTTTAACAATTTATCA) and SD sequence (SD2:GG, SD4:GAGG, SD6:AGGAGG, SD8:TAAGGAGG).<br />
We constructed SD8 from synthetic oligos (forward:SD8-f, reverse:SD8-r).<br />
We constructed SD2, SD4, SD6 by PCR (forward:EX-f, reverse:SD2-r, SD4-r, SD6-r, template:SD8).<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2013/a/ab/HokkaidoU_RBS_methods1_800.png"><br />
<div>fig.1: oligos; RED: enhancer sequence, BLUE: SD sequence.</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/9/9e/HokkaidoU_RBS_methods2_400.png"><br />
<div>fig.2: RBS construction</div><br />
</div><br />
<br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2013/1/1a/HokkaidoU2013_RBS_methods3revision_400.png"><br />
<div>fig.3: our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<h2>Assay</h2><br />
<p><br />
We ligated TetR repressible promoter (pTet), each of the new RBSs', LacZ&alpha; and double terminator.<br />
Using this construct we performed &beta;-Galactosidase assay.<br />
</p><br />
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{{Team:HokkaidoU_Japan/footer}}</div>Baraohttp://2013.igem.org/File:HokkaidoU2013_RBS_methods3revision_400.pngFile:HokkaidoU2013 RBS methods3revision 400.png2013-10-16T16:46:14Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_optimization_fig8slecat.pngFile:HokkaidoU2013 optimization fig8slecat.png2013-09-28T03:23:55Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Modeling_fig3_800.pngFile:HokkaidoU2013 promoter Modeling fig3 800.png2013-09-28T01:25:24Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Result-fig5.pngFile:HokkaidoU2013 promoter Result-fig5.png2013-09-28T01:13:59Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Result-fig4.pngFile:HokkaidoU2013 promoter Result-fig4.png2013-09-28T01:13:01Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Result-fig2.pngFile:HokkaidoU2013 promoter Result-fig2.png2013-09-28T01:12:04Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Result-fig1.pngFile:HokkaidoU2013 promoter Result-fig1.png2013-09-28T01:10:37Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Method-fig4.pngFile:HokkaidoU2013 promoter Method-fig4.png2013-09-28T00:25:08Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Method-fig3.pngFile:HokkaidoU2013 promoter Method-fig3.png2013-09-28T00:24:10Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Method-fig2.pngFile:HokkaidoU2013 promoter Method-fig2.png2013-09-28T00:23:02Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Method-fig1.pngFile:HokkaidoU2013 promoter Method-fig1.png2013-09-28T00:21:21Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Modeling_fig5.pngFile:HokkaidoU2013 promoter Modeling fig5.png2013-09-28T00:16:25Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Modeling_fig4.pngFile:HokkaidoU2013 promoter Modeling fig4.png2013-09-28T00:15:01Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Modeling_fig3.pngFile:HokkaidoU2013 promoter Modeling fig3.png2013-09-28T00:13:50Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Modeling_fig2.pngFile:HokkaidoU2013 promoter Modeling fig2.png2013-09-28T00:12:39Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Modeling_fig1.pngFile:HokkaidoU2013 promoter Modeling fig1.png2013-09-28T00:11:25Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_promoter_Background_fig4.pngFile:HokkaidoU2013 promoter Background fig4.png2013-09-28T00:07:32Z<p>Barao: </p>
<hr />
<div></div>Baraohttp://2013.igem.org/File:HokkaidoU2013_Promoter_background_fig3_new_800.pngFile:HokkaidoU2013 Promoter background fig3 new 800.png2013-09-28T00:04:30Z<p>Barao: </p>
<hr />
<div></div>Barao