http://2014.igem.org/wiki/index.php?title=Special:Contributions/TaKeZo&feed=atom&limit=50&target=TaKeZo&year=&month=2014.igem.org - User contributions [en]2024-03-28T21:26:14ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T03:49:58Z<p>TaKeZo: </p>
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Anti-Sense B0034<br />
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Methods</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stemmed anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; anti-sense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2 ml of LB medium (until the turbidity at OD<sub>600</sub> reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2 ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18 h in M9ZB medium.</p><br />
<br />
<h3>Details of pHN1257 vector</h3><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). Smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). Smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From these experiments, we were not able to confirm whether asRNA worked by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.</p><br />
<br />
<h3>Discussion</h3><br />
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.<br />
<ol><br />
<p><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works on B0034 construct, but it was not functional on H-stem system. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Anti-sense was not expressed sufficiently.</li><br />
From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA expression were not sufficient.<br />
We confirmed the existence of asB0034 by sequencing the vector, though we did not confirm about asB0032. The reason is the difficulty to sequencing of DNA which has stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays we cultured several colonies derived from same origin of plasmids. However, the expression of mRFP showed gap between colonies despite of same level of turbidity. Therefore, because of changes of copy number of target gene, expression of target genes weren’t consistent, and then we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<ol><br />
<p><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of asRNA, we showed the expression can be induced using IPTG.<br />
We will anti-sense works even if copy number is unstable by comparing mRNA of target gene with / without anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce asRNA by IPTG easily, we have used M9ZB medium. However the medium includes glucose, IPTG induction may be too late. We will try to use LB medium to enable IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select plasmids with higher copy number. We will use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We showed asRNA works in case using Nakashima’s stem. On the other hand, in case of H-stem system, we could confirm only transcription of antisense but we could not get a proof that antisense worked. We want to show some results in the presentation at Boston by re-thinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/LengthTeam:HokkaidoU Japan/Projects/Length2014-10-18T03:46:03Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p><br />
Using anti-sense RNA (asRNA) is one of the methods to repress gene expression.<br />
It is known that the length of anti-sense sequence is related to its repression efficiency (N. Nakashima <i>et al.</i>, 2006<sup><a href="#cite-1">[1]</a></sup>), but the details of the relation are still unclear. In this project, we made different lengths of anti-sense sequence (Fig. 1). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/HokkaidoU_length_Overveiw2.png"><br />
<div>Fig. 1 Each anti-sense represses mRNA.</div><br />
</div><br />
<br />
<p><br />
Our experiments will be a clue for other iGEMers who want to design their own anti-sense sequence.</p><br />
<br />
<h1>Introduction</h1><br />
<br />
<p><br />
In repressing gene by anti-sense RNA, it is important to determine the length of anti-sense. However, it is difficult to do it. Theoretically, if the length is too long, it doesn’t repress target RNA effectively. The reason is because the RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them also gets low. However, too short asRNA also has some problems. The short anti-sense cannot bind to the specific part of mRNA because it has too short complementary sequences of target RNA. In the industrial and academic fields, people hope to use anti-sense that has suitable repression efficiency. For example, you can create knock down recombinant organisms easily by using strong anti-sense, and in iGEM, you can make bio-devices which have a complicated gene network and require fine-tuned gene expression. Gene expression is not only ON or OFF. As stated above, each case needs each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is known that this method is difficult.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/HokkaidoU_length_Gifgifgifff.gif"><br />
<div>Fig. 2 Determining a repression region is difficult.</div><br />
</div><br />
<br />
<p><br />
In this project, we made many kinds of anti-sense which repress mRFP, but these anti-sense constructs themselves are not useful for you. However, in our method, you can create the anti-sense that you desire. We expect this project will help you decide anti-sense sequences. We’d like to tell scientists and iGEMers how easy and accurate to repress the gene expression by anti-sense RNA.</p><br />
<br />
<br />
<h1 style="font-size:43px;" id="Method">Methods</h1><br />
<p><br />
We made anti-sense constructs that repress mRFP. The target construct are composed of P<sub>tet</sub> (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>), B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>), mRFP (<a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>) and double terminator (<a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015</a><br />
).<br />
<br />
<br />
Insert fragments were synthesized based on BioBrick by PCR. <br />
As forward primer, ”XhoI-P<sub>tet</sub> (-10)”was used for making all fragments. The primer binds to -10 region of P<sub>tet</sub>, and its end has XhoI restriction enzyme site. To change the downstream seqeunce, each reverse primer is designed differently (as90 NcoI, as120 NcoI) (Fig. 3). These primers bind to each specific part of mRFP, and their ends have NcoI restriction enzyme site. By that way, we can get various length of insert fragments, as90 and as120. As90 is the anti-sense that covers 90 bp of mRNA, and as120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Of course, the edges of insert fragments have restriction enzymes XhoI, NcoI sites. <br />
<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/e/ee/HokkaidoU_length_Figfig3.png"><br />
<div>Fig. 3 Synthesizing insert fragments by PCR</div><br />
</div><br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_Method3.png"><br />
<div>Fig. 4 Ligate the insert fragment with H-stem vector</div><br />
</div><br />
<p><br />
<br />
<p><br />
After we finished synthesizing insert fragments, we inserted them into our H-stem vector (anti-sense expression vector <a href="http://parts.igem.org/Part:BBa_K1524100">BBa_K1524100</a>) by XhoI and NcoI. <br />
<br />
Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector in other experiment (as30 (<a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a>) and as60 (<a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem system</a>)). We performed repression experiment by using their 4 anti-sense constructs.<br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<h2>How to assay</h2><br />
<p><br />
We performed RT-PCR to confirm the transcription of anti-sense RNA (asRNA) constructs.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in 4 mL LB medium for 16 hours.</li><br />
<li>Centrifuged the 4 mL of culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<h1 id="Results">Results</h1><br />
<p><br />
Though we measured absorbance of 260 nm about cDNA, we could not get any cDNA. After RNA extraction, we confirmed absorbance of 260 nm (this is the absorbance of nucleic acid). However, after RT-PCR of that products, we could not confirm the existence of nucleic acid.<br />
<br />
<p><br />
It is presumable that there are some problems in RNA extraction. First, we might have lost RNA during the experiment operation. RNA is degraded easily than DNA because generally RNase can be easily contaminated.</p><br />
<br />
<p><br />
Secondly, the incomplete deactivation of DNase can be considered to be the reason of our failure. If DNase was not deactivated sufficiently, it will result in the degradation of cDNA produced in RT-PCR.</p><br />
<br />
<p><br />
Though we could not make it by wiki freeze, we are going to retry this experiment.</p><br />
<br />
<br />
<br />
<br />
<h1 id="Conclusion">Developmental experiment</h1><br />
<br />
<p><br />
We theoretically estimated that the repression efficiency of asRNA is related to its length. To find the optimum length, we must design many lengths of asRNA, and measure their efficiency. However, that takes a lot of time and labor. So, as a future work, we propose an efficient method to synthesize various length of anti-sense.</p><br />
<br />
<h2>Methods</h2><br />
<br />
<h3>Preparation for randomizing</h3><br />
<br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/HokkaidoU_length_Random1.png"><br />
<div>Fig. 5 PrePCR for randomizing</div><br />
</div><br />
<p><br />
<!<br />
<p><br />
Before randomizing, we have to perform some steps to make the effective anti-sense fragments.<br />
First, we performed PCR on mRFP construct. We call this step "prePCR". We used below primers.</p><br />
<br />
<ul style="padding-left:50px; margin:20px 0;"><br />
<li>XhoI-P<sub>tet</sub> (-10)</li><br />
<li>mRFP 400 down</li><br />
</ul><br />
<br />
XhoI-P<sub>tet</sub> (-10) is a primer that binds to -10 region of P<sub>tet</sub> (BBa_R0040), and its 5’contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). Because it doesn’t contain -35 region, DNA synthesizing starts from P<sub>tet</sub>’s -10 region and PCR products don’t contain a functional part as promoter.<br />
<br><br />
mRFP 400 down is a primer that binds to mRFP (BBa_E1010) 400 bp downstream. <br />
<br><br />
PCR products that are amplified by these 2 primes showed in Fig. 5.<br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<br />
<p><br />
Through this step, we can get insert fragments containing SD (Shine-Dargalno) sequence and start codon that are important to effictive repression.</p><br />
<br />
<h3>Randomizing</h3><br />
<p><br />
For this method, we designed two primers shown below. The second primer is a "random primer" which can bind with any sequence on the template DNA. By using these primers, we can synthesize various lengths of anti-sense. <br />
<br />
<ul style="padding-left:50px; margin:20px 0;"><br />
<li>XhoI-P<sub>tet</sub> (-10)</li><br />
<li>NcoI-NNNNNN</li><br />
</ul><br />
</p><br />
<br />
<p><br />
However, the random primer only has 6 nt of region that anneals with the template DNA. Eventually, the Tm value of this primer gets extremely low. This causes some difficulties to PCR. As a solution, we have to take three unusual steps for PCR. <br />
<br><br />
First, to single-strandize the template DNA, putting it at 95&deg;C / for 3 min and then putting it in 0&deg;C water. By this procedure, the template DNA will be kept denatured in low temperature. <br />
<br>Second, to anneal the primer and amplify the sequences in low temperature, we use "Klenow Fragment", a polymerase whose optimum temperature is 37&deg;C. In this step, we can not amplify fragments, but can make templates of various lengths. <br />
<br>In the third step, we amplify the templates we mentioned above at 68&deg;C just like the usual PCR. </p><br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/HokkaidoU_length_Random3.png"><br />
<div>Fig. 6 How to make random anti-sense</div><br />
</div><br />
<br />
<p><br />
By these methods, you can get various lengths of asRNA of target gene. These fragments are all in one mixture of PCR products. Thus, by digesting, ligating the fragments into a vector and transforming them to cells, you can get various colonies with different lengths of anti-sense. The colony with the optimum length of anti-sense must be on the plate!</p><br />
<br />
<h2>Results</h2><br />
<p><br />
We carried out this plan into practice. We used mRFP as the target gene. After prePCR, we performed PCR by following the steps we described above. The protocol of PCR is shown below. <br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/HokkaidoU_length_Last_recipe.png"><br />
<div>Fig. 7 Randomizing protocol</div><br />
</div><br />
<br />
<div class="clearfix"><br />
</div><br />
<br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/4/4c/HokkaidoU_length_Protocols.png"><br />
<div>Fig. 8 Results of randomizing</div><br />
</div><br />
<p><br />
<!<br />
<p><br />
We performed an experiment in two protocols, A and B. The annealing temperature and PCR cycles are different beteween A and B. We inserted them into vectors and transformed them to cells. We got some colonies in both plates! More colonies appeared in Protocol B than Protocol A. After that, we performed colony PCR. However, we could not get the result of colony PCR.<br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<br />
<h2>Discussion</h2><br />
<p><br />
Getting this result that the colony PCR had failed, we stopped this project. However, recently, we realized that the stem sequences have difficuties in PCR (according to Mr. Nakashima). Therefore, we cannot conclude that this project have completely failed.</p><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18h in M9ZB medium.</p><br />
<br />
<h3>Details of pHN1257 vector</h3><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From these experiments, we were not able to confirm whether asRNA worked by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.</p><br />
<br />
<h3>Discussion</h3><br />
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.<br />
<ol><br />
<p><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works on B0034 construct, but it was not functional on H-stem system. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense was not expressed sufficiently.</li><br />
From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA expression were not sufficient.<br />
We confirmed the existence of antisense B0034 by sequencing the vector, though we did not confirm about antisense B0032. The reason is the difficulty to sequencing of DNA which has stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays we cultured several colonies derived from same origin of plasmids. However, the expression of mRFP showed gap between colonies despite of same level of turbidity. Therefore, because of changes of copy number of target gene, expression of target genes weren’t consistent, and then we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<ol><br />
<p><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of asRNA, we showed the expression can be induced using IPTG.<br />
We will anti-sense works even if copy number is unstable by comparing mRNA of target gene with / without anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce asRNA by IPTG easily, we have used M9ZB medium. However the medium includes glucose, IPTG induction may be too late. We will try to use LB medium to enable IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select plasmids with higher copy number. We will use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We showed asRNA works in case using Nakashima’s stem. On the other hand, in case of H-stem system, we could confirm only transcription of antisense but we could not get a proof that antisense worked. We want to show some results in the presentation at Boston by re-thinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T03:22:27Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18h in M9ZB medium.</p><br />
<br />
<h3>Details of pHN1257 vector</h3><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From these experiments, we were not able to confirm whether asRNA worked by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.</p><br />
<br />
<h3>Discussion</h3><br />
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.<br />
<ol><br />
<p><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works on B0034 construct, but it was not functional on H-stem system. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense was not expressed sufficiently.</li><br />
From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA expression were not sufficient.<br />
We confirmed the existence of antisense B0034 by sequencing the vector, though we did not confirm about antisense B0032. The reason is the difficulty to sequencing of DNA which has stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<ol><br />
<p><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of asRNA, we showed the expression can be induced using IPTG.<br />
We will anti-sense works even if copy number is unstable by comparing mRNA of target gene with / without anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce asRNA by IPTG easily, we have used M9ZB medium. However the medium includes glucose, IPTG induction may be too late. We will try to use LB medium to enable IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select plasmids with higher copy number. We will use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We showed asRNA works in case using Nakashima’s stem. On the other hand, in case of H-stem system, we could confirm only transcription of antisense but we could not get a proof that antisense worked. We want to show some results in the presentation at Boston by re-thinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T03:13:44Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18h in M9ZB medium.</p><br />
<br />
<h2>Details of pHN1257 vector</h2><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From these experiments, we were not able to confirm whether asRNA worked by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.</p><br />
<br />
<h2>Discussion</h2><br />
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.<br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works on B0034 construct, but it was not functional on H-stem system. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense was not expressed sufficiently.</li><br />
From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA expression were not sufficient.<br />
We confirmed the existence of antisense B0034 by sequencing the vector, though we did not confirm about antisense B0032. The reason is the difficulty to sequencing of DNA which has stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T03:08:01Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18h in M9ZB medium.</p><br />
<br />
<h2>Details of pHN1257 vector</h2><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From these experiments, we were not able to confirm whether asRNA worked by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.</p><br />
<br />
<h2>Discussion</h2><br />
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.<br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense was not expressed sufficiently.</li><br />
From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA were not expressed sufficiently.<br />
We confirmed the existence of antisense B0034 by sequencing the vector, though we did not confirm about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T03:01:03Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18h in M9ZB medium.</p><br />
<br />
<h2>Details of pHN1257 vector</h2><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From these experiments, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 worked.</p><br />
<br />
<h2>Discussion</h2><br />
We are assuming three possible causes of that H-stem system made no difference between the cases with and withou IPTG induction.<br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.10, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not confirm about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T02:25:09Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>The targets of these asRNA are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated a colony of transformed bacteria in 2ml of LB medium (until the turbidity at OD600 reached 0.1).</li><br />
<li>Retrieved the bacteria and cultivated them in 2ml of M9ZB medium</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Results</h1><br />
<p>We assessed the efficiency to suppress expression of two kinds of asRNA family: one has PT structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of <i>E. coli</i>:</p><br />
<ul><br />
<li>target B0034 + anti-sense B0034 </li><br />
<li>target B0034 + anti-sense B0032 </li><br />
<li>target B0032 + anti-sense B0034 </li><br />
<li>target B0032 + anti-sense B0032 </li><br />
</ul><br />
<p>We examined each sample with / without IPTG induction.</p><br />
<br />
<h2>Nakashima's stem</h2><br />
<p><br />
We assessed whether asRNA with Nakashima's stem work. We used Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample <i>E. coli</i> were cultivated for 18h in M9ZB medium.</p><br />
<br />
<h2>Details of pHN1257 vector</h2><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) structure. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT sites. The vector resistance is Kanamycin. Also, it has IPTG inducible promoter, P<sub>trc</sub>. Copy number is 30 (reprication origin is pSC101). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h2>H-stem</h2><br />
<p>We inserted anti-sense between H-stem insted of PT structure and assayed their efficiency.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 worked.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.10, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not confirm about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T01:44:01Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>This is how to synthesize anti-sense constructs. Their targets are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in LB medium.</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We assessed whether anti-sense B0034 works as asRNA by using Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1.</p><br />
<br />
<h2>Details of pHN1257 vector</h2><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) design. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT. The vector resistance is Kanamycin, has IPTG induce promoter, P<sub>trc</sub> and 30 copy numbers (reprication origin is pSC101). </p><br />
<br />
<br />
<br />
<br />
<h3>Our samples are following</h3><br />
<ul><br />
<li>target B0034+ anti-sense B0034 </li><br />
<li>target B0034+ anti-sense B0032 </li><br />
<li>target B0032+ anti-sense B0034 </li><br />
<li>target B0032+ anti-sense B0032 </li><br />
</ul><br />
<p>We examined each samples with / without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP as a target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene were used for assay.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in 2 mL LB culture for about 18 hours</li><br />
<li>Controled turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>Cultivated the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>Measured fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 IPTG(+) / IPTG(-) ratio in fluorescence strength. smaller values mean that the target gene was suppressed by IPTG induction.</div><br />
</div><br />
<br />
<p>Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). smaller values indicate that the mRFP gene was suppressed because of asRNA, induced by IPTG.<br />
In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work.<br />
From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.</p><br />
<br />
<h1>Results</h1><br />
<p>We inserted anti-sense into H-stem and assayed them to make anti-sense working in not only Nakashima’s stem but on our own H-stem.<br />
All anti-senses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples were following</h3><br />
<ul><br />
<li>target B0034+ anti-sense B0034</li><br />
<li>target B0034+ anti-sense B0032 </li><br />
<li>target B0032+ anti-sense B0034 </li><br />
<li>target B0032+ anti-sense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>Methods for assay</h3><br />
We selected mRFP as a target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in 4 mL LB culture for about 22 hours</li><br />
<li>Controled turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>cultivated the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>Measured fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 worked.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.10, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not confirm about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-18T01:23:46Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_AsB0034_fig1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
As a solution, we decided to design a general asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/25/HokkaidoU_length_AsB0034_fig02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
To achieve this, we constructed an asRNA for RBS. <br />
For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects.<br />
Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (<a href="http://parts.igem.org/Part:BBa_K1524107">BBa_K1524107</a>)".<br />
By this part, iGEMers can repress any target gene that is synthesised downstream B0034.<br />
<br />
</p><br />
<p></p><br />
<p><br />
We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (<a href="http://parts.igem.org/Part:BBa_K1524108">BBa_K1524108</a>)".<br />
You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.<br />
</p><br />
<p><br />
You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/2a/HokkaidoU_asB0034_Anti-senseB0034.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">Method</h1><br />
<p>This is how to synthesize anti-sense constructs. Their targets are B0034 (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a>) and B0032 (<a href="http://parts.igem.org/Part:BBa_B0032">BBa_B0032</a>). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 4 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f0/HokkaidoU_B0034_AsB0034asB0032.png"><br />
<div>Fig. 7 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 8 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in LB medium.</li><br />
<li>Centrifuged the culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Removed the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Performed RT-PCR</li><br />
<li>Measured absorbance of 260 nm about cDNA.</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 9 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We assessed whether anti-sense B0034 works as asRNA by using Nakashima’s plasmid (pHN1257<sup><a href="#cite-1">[1]</a></sup><sup><a href="#cite-1">[2]</a></sup>) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1.</p><br />
<br />
<h2>Details of pHN1257 vector</h2><br />
<p>This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) design. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT. The vector resistance is Kanamycin, has IPTG induce promoter, P<sub>trc</sub> and 30 copy numbers (reprication origin is pSC101). </p><br />
<br />
<br />
<br />
<br />
<h3>Our samples are following</h3><br />
<ul><br />
<li>target B0034+ anti-sense B0034 </li><br />
<li>target B0034+ anti-sense B0032 </li><br />
<li>target B0032+ anti-sense B0034 </li><br />
<li>target B0032+ anti-sense B0032 </li><br />
</ul><br />
<p>We examined each samples with / without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP as a target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene were used for assay.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in 2 mL LB culture for about 18 hours</li><br />
<li>Controled turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>Cultivated the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>Measured fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 10 Fluorescence strength of each sample with / without IPTG</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 11 Rate of IPTG(+) and IPTG(-). <br />
Numeric became small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 11, we were able to see the translation of asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 were down regulated the expression 40%, and asB0032 showed the regulation of 80%. Doc. Nakashima gained a regulation of 78%, so we were able to get the same result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted anti-sense into H-stem and assayed them to make anti-sense working in not only Nakashima’s stem but on our own H-stem.<br />
All anti-senses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul style="list-style-image:none; list-style-type:none;"><br />
<li>target B0034+ anti-sense B0034</li><br />
<li>target B0034+ anti-sense B0032 </li><br />
<li>target B0032+ anti-sense B0034 </li><br />
<li>target B0032+ anti-sense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>Methods for assay</h3><br />
We selected mRFP as a target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>Cultivated the colony in 4 mL LB culture for about 22 hours</li><br />
<li>Controled turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>cultivated the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>Measured fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 12 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 13 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 14 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 worked.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 12, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.10, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not confirm about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.10 and 12, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 15 Rate of RBS used BioBrick parts</div><br />
</div><br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.</p><br />
<div class="clearfix"></div><br />
<br />
<br><br />
<br><br />
<br />
<h3>Reference</h3><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N. Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
<br />
<li id="cite-2">N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of <i>Escherichia coli</i>. Nucleic Acids Res 37: 15 e103</li><br />
</ol><br />
<br />
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</html></div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-17T18:59:33Z<p>TaKeZo: </p>
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Anti-Sense B0034<br />
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_project_antisenseB0034_overview01_800_1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
Therefore, we decided to design an universal asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/HokkaidoU_antisenseB0034_overview02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
As a solution, we decided to make a new part <br />
<br />
We found that anti-sense RBS (B0034) fragment, which is used by some iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.<br />
</p><br />
<p></p><br />
<p><br />
We also synthesized anti-sense B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.<br />
</p><br />
<p><br />
Specific anti-sense RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. All you have to do is to add our anti-sense fragment to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">How to synthesize anti-sense constructs</h1><br />
<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 2 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense conplex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"><br />
<div>Fig. 4 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 5 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li><br />
<li>To control turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 9 hour</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034 </li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 2 mL LB culture for about 18 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 1 Value of fluorescence of IPTG with and without each sample</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 2 Rate of IPTG(+) and IPTG(-)<br />
Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem.<br />
All antisenses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034</li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 22 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 3 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 4 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 5 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 6 Rate of RBS used BioBrick parts</div><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-17T18:59:08Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_project_antisenseB0034_overview01_800_1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
Therefore, we decided to design an universal asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/HokkaidoU_antisenseB0034_overview02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
As a solution, we decided to make a new part <br />
<br />
We found that anti-sense RBS (B0034) fragment, which is used by some iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.<br />
</p><br />
<p></p><br />
<p><br />
We also synthesized anti-sense B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.<br />
</p><br />
<p><br />
Specific anti-sense RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. All you have to do is to add our anti-sense fragment to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">How to synthesize anti-sense constructs</h1><br />
<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 2 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense conplex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"><br />
<div>Fig. 4 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 5 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li><br />
<li>To control turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 9 hour</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034 </li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 2 mL LB culture for about 18 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 1 Value of fluorescence of IPTG with and without each sample</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 2 Rate of IPTG(+) and IPTG(-)<br />
Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem.<br />
All antisenses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034</li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 22 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 3 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 4 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 5 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
<br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 6 Rate of RBS used BioBrick parts</div><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-17T18:58:25Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_project_antisenseB0034_overview01_800_1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
Therefore, we decided to design an universal asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/HokkaidoU_antisenseB0034_overview02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
As a solution, we decided to make a new part <br />
<br />
We found that anti-sense RBS (B0034) fragment, which is used by some iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.<br />
</p><br />
<p></p><br />
<p><br />
We also synthesized anti-sense B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.<br />
</p><br />
<p><br />
Specific anti-sense RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. All you have to do is to add our anti-sense fragment to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">How to synthesize anti-sense constructs</h1><br />
<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 2 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense conplex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"><br />
<div>Fig. 4 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 5 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li><br />
<li>To control turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 9 hour</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034 </li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 2 mL LB culture for about 18 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 1 Value of fluorescence of IPTG with and without each sample</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 2 Rate of IPTG(+) and IPTG(-)<br />
Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem.<br />
All antisenses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034</li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 22 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 3 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 4 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 5 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
</ol><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 6 Rate of RBS used BioBrick parts</div><br />
</div><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-17T18:57:42Z<p>TaKeZo: Undo revision 342890 by TaKeZo (talk)</p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_project_antisenseB0034_overview01_800_1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
Therefore, we decided to design an universal asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/HokkaidoU_antisenseB0034_overview02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
As a solution, we decided to make a new part <br />
<br />
We found that anti-sense RBS (B0034) fragment, which is used by some iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.<br />
</p><br />
<p></p><br />
<p><br />
We also synthesized anti-sense B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.<br />
</p><br />
<p><br />
Specific anti-sense RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. All you have to do is to add our anti-sense fragment to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">How to synthesize anti-sense constructs</h1><br />
<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 2 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense conplex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"><br />
<div>Fig. 4 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 5 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li><br />
<li>To control turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 9 hour</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034 </li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 2 mL LB culture for about 18 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 1 Value of fluorescence of IPTG with and without each sample</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 2 Rate of IPTG(+) and IPTG(-)<br />
Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem.<br />
All antisenses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034</li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 22 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 3 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 4 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 5 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 6 Rate of RBS used BioBrick parts</div><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-17T18:56:50Z<p>TaKeZo: </p>
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA (asRNA) is studied actively over the world. <br />
asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA.<br />
It is a labor to design efficient asRNA for every target gene you want to repress. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bb/HokkaidoU_project_antisenseB0034_overview01_800_1.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p><br />
Therefore, we decided to design an universal asRNA which could repress various target genes.<br />
<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/bc/HokkaidoU_antisenseB0034_overview02.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p><br />
As a solution, we decided to make a new part <br />
<br />
We found that anti-sense RBS (B0034) fragment, which is used by some iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.<br />
</p><br />
<p></p><br />
<p><br />
We also synthesized anti-sense B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.<br />
</p><br />
<p><br />
Specific anti-sense RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. All you have to do is to add our anti-sense fragment to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">How to synthesize anti-sense constructs</h1><br />
<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 2 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense conplex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"><br />
<div>Fig. 4 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 5 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li><br />
<li>To control turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 9 hour</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034 </li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 2 mL LB culture for about 18 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 1 Value of fluorescence of IPTG with and without each sample</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 2 Rate of IPTG(+) and IPTG(-)<br />
Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem.<br />
All antisenses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034</li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 22 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 3 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 4 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 5 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.</p><br />
<br />
<h2>Discussion</h2><br />
<dl style="list-style: decimal"><br />
<tt>Anti-sense was not induced by IPTG; it is leaking.</tt><br />
</td>Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</td><br />
<br />
<tt>Antisense did not show any expression</tt><br />
<td>Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</td><br />
<tt>Instability of copy number of target gene</tt><br />
<td>Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</td><br />
</dl><br />
<br />
<br />
<h1>Improvement</h1><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 6 Rate of RBS used BioBrick parts</div><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:47:03Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:44:57Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:44:09Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:43:26Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:42:26Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:41:35Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:40:58Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:38:15Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:37:54Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:37:14Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/PartsTeam:HokkaidoU Japan/Parts2014-10-17T18:35:29Z<p>TaKeZo: </p>
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/About_UsTeam:HokkaidoU Japan/About Us2014-10-17T18:31:02Z<p>TaKeZo: </p>
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About Us<br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
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<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us">About Us</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
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<h1 id="Acknowledgements">Acknowledgements</h1><br />
<h2>Sponsors</h2><br />
<div class="article"><br />
<ul id="sponsor-list"><br />
<li><a href="http://www.aminoup.co.jp/e/"><img src="https://static.igem.org/mediawiki/2014/a/a2/HokkaidoU_sponsorlogo_Aminoup.png"></a></li><br />
<li><a href="http://www.tomoechan.jp/"><img src="https://static.igem.org/mediawiki/2014/f/f2/HokkaidoU_sponsorlogo_Fukuyamaziyouzou.png"></a></li><br />
<li><a href="http://www.hokudai.ac.jp/"><img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_sponsorlogo_Hokudai.png"></a></li><br />
<li><a href="http://www.cosmobio.co.jp/index_e.asp"><img src="https://static.igem.org/mediawiki/2014/f/fa/HokkaidoU_sponsorlogo_Cosmobio.png"></a></li><br />
<li><a href="http://mendel-science.com/"><img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_sponsorlogo_Menderukobo.png"></a></li><br />
<li><a href="http://www.toyobo-global.com/"><img src="https://static.igem.org/mediawiki/2014/b/b5/HokkaidoU_sponsorlogo_Toyobo.png"></a></li><br />
</ul><br />
</div><br />
<br />
<br />
<br />
<h2>Collaborator</h2><br />
<h3>iGEM Japan</h3><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3f/HokkaidoU_Genetic_Learned_Society.jpg"><br />
</div><br />
<p><br />
We attended the 86th Genetic Society of Japan and discuss with 8 Japanese iGEM teams (Gifu, HokkaidoU, Kyoto, Nagahama, Osaka, TMU-tokyo, Tokyo-tech, UT-tokyo).<br />
<br />
<h2>Special thanks</h2><br />
<h3>Nobutaka Nakashima</h3><br />
<h3>Tomohiro Tamura</h3><br />
<br />
<br />
<br />
</p> <br />
<h1 id="Team">Our Team</h1><br />
<div class="fig fig800"><br />
<img id="team-picture" src="https://static.igem.org/mediawiki/2014/4/47/IGEM_HokkaidoU_Japan_Team.jpg" usemap="#Map"><br />
<map name="Map"><br />
<area class="member-click" shape="rect" coords="483,250,527,297 " href="#instructor" ><br />
<area class="member-click" shape="rect" coords="268,189,321,238 " href="#liu" ><br />
<area class="member-click" shape="rect" coords="545,175,594,224" href="#saitoh" ><br />
<area class="member-click" shape="rect" coords="77,95,119,143" href="#kawahata" ><br />
<area class="member-click" shape="rect" coords="232,243,280,289" href="#hana" ><br />
<area class="member-click" shape="rect" coords="310,92,354,148" href="#hana" ><br />
<area class="member-click" shape="rect" coords="368,124,415,174" href="#ayano" ><br />
<area class="member-click" shape="rect" coords="448,105,500,151" href="#osamu" ><br />
<area class="member-click" shape="rect" coords="161,236,204,295" href="#tamaro" ><br />
<area class="member-click" shape="rect" coords="95,236,140,285" href="#tori" ><br />
<area class="member-click" shape="rect" coords="113,187,155,243" href="#tamaro" ><br />
<area class="member-click" shape="rect" coords="312,248,360,290" href="#tori" ><br />
<area class="member-click" shape="rect" coords="342,197,385,246" href="#osamu" ><br />
<area class="member-click" shape="rect" coords="" href="#" ><br />
<area class="member-click" shape="rect" coords="" href="#" ><br />
</map><br />
</div><br />
<br />
<div class="member"><br />
<div class="member-header" id="instructor"><br />
<h2 class="member-title">Instructors</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="ymzk"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2013/4/43/HokkaidoU_2013_Ymzk.png"><br />
<div class="overlay"><br />
<h3><br />
Ken-ici YAMAZAKI<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Group of Environmental Molecular Biology, Section of Environmental Biology, Faculty of Environmental Earth Science<br><br />
<!--(<a href="http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html">Yamazaki Lab</a>)--><br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="sone"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/0/0e/HokkaidoU_Member_sone.png"><br />
<div class="overlay"><br />
<h3><br />
Teruo SONE<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Group of Applied Mycology, Section of Molecular Biology, Faculty of Agriculture<br><br />
<!--(<a href="http://www.agr.hokudai.ac.jp/oukin/index.html">Applied Mycology Lab</a>)--><br />
</p><br />
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</div><br />
</li><br />
<div class="clearfix"></div><br />
</ul><br />
</div><br />
</div><br />
<br />
<br />
<div class="member"><br />
<div class="member-header2" id="undergrads"><br />
<h2 class="member-title">Team Members</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="hana"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/6/60/HokkaidoU_Member_shimoyama_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Hana SHIMOYAMA<br />
</h3><br />
<div class="overlay-content"><br />
<p>Team Leader</p><br />
<p><br />
Faculty: Agriculture Plant physiology (B3)<br><br />
My hobby is cultivating vegetable. <br> I like chili food!! I work in soup-curry shop.<br><br />
I like iGEM,<i> E. coli</i> and team members!!!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="ryotaro"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_Member_nouda_off.JPG"><br />
<div class="overlay"><br />
<h3><br />
Ryotaro NOUDA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Experiment Leader<br />
</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I do what I can do. In other matters, I depend on you!<br><br />
I want to study virology, please teach me it!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="ayano"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/2b/HokkaidoU_Member_hata_off.JPG"><br />
<div class="overlay"><br />
<h3><br />
Ayano HATA<br />
</h3><br />
<div class="overlay-content"><br />
<p>Designer / Charge of Human practice</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I always do what I want to do freely. No one can bother me anyway.<br><br />
I treasure my all experiences because everything I meet makes me new. <br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="naoya"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/3/3b/HokkaidoU_Member_matsumura_on.jpg"><br />
<div class="overlay"><br />
<h3><br />
Naoya MATSUMURA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Modeling Supervisor<br />
</p><br />
<p><br />
Faculty: Medicine (B3)<br><br />
In fact, I love physics better than biology. <br><br />
I hope the mystery of life would be described as an elegant physical equation!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="osamu"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/df/HokkaidoU_Member_horiguchi_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Osamu HORIGUCHI<br />
</h3><br />
<div class="overlay-content"><br />
<p>wiki Supervisor</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I like reptiles, especially snakes and lizards. <br><br />
If I will be born again, I want to be born as Raptor or something that can fly.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="eri"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/26/HokkaidoU_Member_mitobe.JPG"><br />
<div class="overlay"><br />
<h3><br />
Eri MITOBE<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Mathematic Girl<br />
</p><br />
<p><br />
Faculty:Science Mathematics (B3)<br><br />
I love sweets!<br><br />
I want to eat delicious sweets in your country.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="tori"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/1/10/HokkaidoU_Member_touri_on.jpg"><br />
<div class="overlay"><br />
<h3><br />
Tori SUZUKI<br />
</h3><br />
<div class="overlay-contenti"><br />
<p>Part Time Job Soldier</p><br />
<p><br />
Faculty: Fisheries Science,Aquaculture Life Science (B2)<br><br />
I love eating,especially foreign cuisine.<br />
So I'm happy if you tell me your country cuisine.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="mami"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/9/99/HokkaidoU_Member_tanaka.jpg"><br />
<div class="overlay"><br />
<h3><br />
Mami TANAKA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Primer Designer<br />
</p><br />
<p><br />
Faculty:Fisheries Science, Aquaculture Life Science (B2)<br><br />
I'm in love with planktons, especially Closteriums.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="tamaro"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/d6/HokkaidoU_Member_tamaro_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Tamaro SAKURAI<br />
</h3><br />
<div class="overlay-content"><br />
<p></p><br />
<p>Faculty:Science Biology (B2)<br>I like sports especially cycle road race and triathlon. <br><br />
I want to study and solve the mechanism that differentiated cell obtains pluripotency.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="sanbon"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_Member_sanbon.jpg"><br />
<div class="overlay"><br />
<h3><br />
Masaya MITSUMOTO<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
self-introduction<br />
</p><br />
<p>Faculty:Science Biology (B2)<br><br />
I like funny anime or movies for example, The Hangover, Johnny English, Spongebob SquarePants etc. So if you have some recommendation, please give me the title of them!</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="hosoki"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/8/89/HokkaidoU_Member_hosoki.jpg"><br />
<div class="overlay"><br />
<h3><br />
Takuya HOSOKI<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
self-introduction<br />
</p><br />
<p>Faculty:Fisheries Science,Aquaculture Life Science (B2)<br><br />
I love fish.Fish love me.</p><br />
</div><br />
</div><br />
</li><br />
<br />
<br />
<li id="saitoh"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/e/e1/HokkaidoU_Member_saitoh.jpg"><br />
<div class="overlay"><br />
<h3><br />
Kensuke SAITOH<br />
</h3><br />
<div class="overlay-content"><br />
<p>Faculty:Science, Biology (B4)<br><br />
I enjoy every day very much since I am stimulated by motivated members!</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="liu"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/a/af/HokkadoU_Member_liu.jpg"><br />
<div class="overlay"><br />
<h3><br />
Yuxiang LIU<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Graduate School of Env.Science<br />
</p><br />
<p><br />
Try something interesting and try something new.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="shehata"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/6/69/IGEM_HokkaidoU_Member_Shehata.jpg"><br />
<div class="overlay"><br />
<h3><br />
Mohamed SHEHATA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Graduate School of Env.Science /<br>Assistant lecturer at Al-Azhar University<br />
</p><br />
<p><br />
I like igem team so much, these students are smart, cooperative, and hard worker, I always learn from them many things.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<div class="clearfix"></div><br />
</ul><br />
</div><br />
</div> <!--Member list--><br />
<br />
<br />
<div class="member"><br />
<div class="member-header3" id="special-thanks"><br />
<h2 class="member-title">Special Thanks</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="itoh"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/4/44/HokkaidoU_Member_Itoh_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Takeshi ITOH<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Wiki Advisor<br />
</p><br />
<p><br />
Faculty: Medicine (B4)<br>I was a wiki Jedi in HokkaidoU last year, and now I came back to this team to pass on my experience and help my apprentices to feel the force of HTML! May the force be with you...<br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="kawahata"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/a/a5/HokkaidoU_Member_kawahata_off.png"><br />
<div class="overlay"><br />
<h3><br />
Ami KAWAHATA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Documents Proofreader<br />
</p><br />
<p>Faculty:Science, Biology (B4)<br>Its my third time to participate in iGEM !<br />
Every year I feel the team growing bigger, and better.<br />
iGEM is a great place to change your life, and your world !<br />
have fun if you can</p><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/About_UsTeam:HokkaidoU Japan/About Us2014-10-17T18:30:30Z<p>TaKeZo: </p>
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About Us<br />
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<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li><br />
</ul><br />
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<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span><br />
<div class="ldd_submenu" style="width:590px; left:-230px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
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<span class="menu-list"><span style="position:relative; top:10px;">About Us</span></span><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us">About Us</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
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<h1 id="Acknowledgements">Acknowledgements</h1><br />
<h2>Sponsors</h2><br />
<div class="article"><br />
<ul id="sponsor-list"><br />
<li><a href="http://www.aminoup.co.jp/e/"><img src="https://static.igem.org/mediawiki/2014/a/a2/HokkaidoU_sponsorlogo_Aminoup.png"></a></li><br />
<li><a href="http://www.tomoechan.jp/"><img src="https://static.igem.org/mediawiki/2014/f/f2/HokkaidoU_sponsorlogo_Fukuyamaziyouzou.png"></a></li><br />
<li><a href="http://www.hokudai.ac.jp/"><img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_sponsorlogo_Hokudai.png"></a></li><br />
<li><a href="http://www.cosmobio.co.jp/index_e.asp"><img src="https://static.igem.org/mediawiki/2014/f/fa/HokkaidoU_sponsorlogo_Cosmobio.png"></a></li><br />
<li><a href="http://mendel-science.com/"><img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_sponsorlogo_Menderukobo.png"></a></li><br />
<li><a href="http://www.toyobo-global.com/"><img src="https://static.igem.org/mediawiki/2014/b/b5/HokkaidoU_sponsorlogo_Toyobo.png"></a></li><br />
</ul><br />
</div><br />
<br />
<br />
<br />
<h2>Collaborator</h2><br />
<h3>iGEM Japan</h3><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3f/HokkaidoU_Genetic_Learned_Society.jpg"><br />
</div><br />
<p><br />
We attended the 86th Genetic Society of Japan and discuss with 8 Japanese iGEM teams (Gifu, HokkaidoU, Kyoto, Nagahama, Osaka, TMU-tokyo, Tokyo-tech, UT-tokyo).<br />
<br />
<h2>Special thanks</h2><br />
<h3>Nobutaka Nakashima</h3><br />
<h3>Tomohiro Tamura</h3><br />
<br />
<br />
<br />
</p> <br />
<h1 id="Team">Our Team</h1><br />
<div class="fig fig800"><br />
<img id="team-picture" src="https://static.igem.org/mediawiki/2014/4/47/IGEM_HokkaidoU_Japan_Team.jpg" usemap="#Map"><br />
<map name="Map"><br />
<area class="member-click" shape="rect" coords="483,250,527,297 " href="#instructor" ><br />
<area class="member-click" shape="rect" coords="268,189,321,238 " href="#liu" ><br />
<area class="member-click" shape="rect" coords="545,175,594,224" href="#saitoh" ><br />
<area class="member-click" shape="rect" coords="77,95,119,143" href="#kawahata" ><br />
<area class="member-click" shape="rect" coords="232,243,280,289" href="#hana" ><br />
<area class="member-click" shape="rect" coords="310,92,354,148" href="#hana" ><br />
<area class="member-click" shape="rect" coords="368,124,415,174" href="#ayano" ><br />
<area class="member-click" shape="rect" coords="448,105,500,151" href="#osamu" ><br />
<area class="member-click" shape="rect" coords="161,236,204,295" href="#tamaro" ><br />
<area class="member-click" shape="rect" coords="95,236,140,285" href="#tori" ><br />
<area class="member-click" shape="rect" coords="113,187,155,243" href="#tamaro" ><br />
<area class="member-click" shape="rect" coords="312,248,360,290" href="#tori" ><br />
<area class="member-click" shape="rect" coords="342,197,385,246" href="#osamu" ><br />
<area class="member-click" shape="rect" coords="" href="#" ><br />
<area class="member-click" shape="rect" coords="" href="#" ><br />
</map><br />
</div><br />
<br />
<div class="member"><br />
<div class="member-header" id="instructor"><br />
<h2 class="member-title">Instructors</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="ymzk"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2013/4/43/HokkaidoU_2013_Ymzk.png"><br />
<div class="overlay"><br />
<h3><br />
Ken-ici YAMAZAKI<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Group of Environmental Molecular Biology, Section of Environmental Biology, Faculty of Environmental Earth Science<br><br />
<!--(<a href="http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html">Yamazaki Lab</a>)--><br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="sone"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/0/0e/HokkaidoU_Member_sone.png"><br />
<div class="overlay"><br />
<h3><br />
Teruo SONE<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Group of Applied Mycology, Section of Molecular Biology, Faculty of Agriculture<br><br />
<!--(<a href="http://www.agr.hokudai.ac.jp/oukin/index.html">Applied Mycology Lab</a>)--><br />
</p><br />
</div><br />
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</li><br />
<div class="clearfix"></div><br />
</ul><br />
</div><br />
</div><br />
<br />
<br />
<div class="member"><br />
<div class="member-header2" id="undergrads"><br />
<h2 class="member-title">Team Members</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="hana"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/6/60/HokkaidoU_Member_shimoyama_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Hana SHIMOYAMA<br />
</h3><br />
<div class="overlay-content"><br />
<p>Team Leader</p><br />
<p><br />
Faculty: Agriculture Plant physiology (B3)<br><br />
My hobby is cultivating vegetable. <br> I like chili food!! I work in soup-curry shop.<br><br />
I like iGEM,<i> E. coli</i> and team members!!!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="ryotaro"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_Member_nouda_off.JPG"><br />
<div class="overlay"><br />
<h3><br />
Ryotaro NOUDA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Experiment Leader<br />
</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I do what I can do. In other matters, I depend on you!<br><br />
I want to study virology, please teach me it!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="ayano"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/2b/HokkaidoU_Member_hata_off.JPG"><br />
<div class="overlay"><br />
<h3><br />
Ayano HATA<br />
</h3><br />
<div class="overlay-content"><br />
<p>Designer / Charge of Human practice</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I always do what I want to do freely. No one can bother me anyway.<br><br />
I treasure my all experiences because everything I meet makes me new. <br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="naoya"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/3/3b/HokkaidoU_Member_matsumura_on.jpg"><br />
<div class="overlay"><br />
<h3><br />
Naoya MATSUMURA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Modeling Supervisor<br />
</p><br />
<p><br />
Faculty: Medicine (B3)<br><br />
In fact, I love physics better than biology. <br><br />
I hope the mystery of life would be described as an elegant physical equation!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="osamu"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/df/HokkaidoU_Member_horiguchi_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Osamu HORIGUCHI<br />
</h3><br />
<div class="overlay-content"><br />
<p>wiki Supervisor</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I like reptiles, especially snakes and lizards. <br><br />
If I will be born again, I want to be born as Raptor or something that can fly.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="eri"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/26/HokkaidoU_Member_mitobe.JPG"><br />
<div class="overlay"><br />
<h3><br />
Eri MITOBE<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Mathematic Girl<br />
</p><br />
<p><br />
Faculty:Science Mathematics (B3)<br><br />
I love sweets!<br><br />
I want to eat delicious sweets in your country.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="tori"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/1/10/HokkaidoU_Member_touri_on.jpg"><br />
<div class="overlay"><br />
<h3><br />
Tori SUZUKI<br />
</h3><br />
<div class="overlay-contenti"><br />
<p>Part Time Job Soldier</p><br />
<p><br />
Faculty: Fisheries Science,Aquaculture Life Science (B2)<br><br />
I love eating,especially foreign cuisine.<br />
So I'm happy if you tell me your country cuisine.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="mami"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/9/99/HokkaidoU_Member_tanaka.jpg"><br />
<div class="overlay"><br />
<h3><br />
Mami TANAKA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Primer Designer<br />
</p><br />
<p><br />
Faculty:Fisheries Science, Aquaculture Life Science (B2)<br><br />
I'm in love with planktons, especially Closteriums.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="tamaro"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/d6/HokkaidoU_Member_tamaro_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Tamaro SAKURAI<br />
</h3><br />
<div class="overlay-content"><br />
<p></p><br />
<p>Faculty:Science Biology (B2)<br>I like sports especially cycle road race and triathlon. <br><br />
I want to study and solve the mechanism that differentiated cell obtains pluripotency.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="sanbon"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_Member_sanbon.jpg"><br />
<div class="overlay"><br />
<h3><br />
Masaya MITSUMOTO<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
self-introduction<br />
</p><br />
<p>Faculty:Science Biology (B2)<br><br />
I like funny anime or movies for example, The Hangover, Johnny English, Spongebob SquarePants etc. So if you have some recommendation, please give me the title of them!</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="hosoki"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/8/89/HokkaidoU_Member_hosoki.jpg"><br />
<div class="overlay"><br />
<h3><br />
Takuya HOSOKI<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
self-introduction<br />
</p><br />
<p>Faculty:Fisheries Science,Aquaculture Life Science (B2)<br><br />
I love fish.Fish love me.</p><br />
</div><br />
</div><br />
</li><br />
<br />
<br />
<li id="saitoh"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/e/e1/HokkaidoU_Member_saitoh.jpg"><br />
<div class="overlay"><br />
<h3><br />
Kensuke SAITOH<br />
</h3><br />
<div class="overlay-content"><br />
<p>Faculty:Science, Biology (B4)<br><br />
I enjoy every day very much since I am stimulated by motivated members!</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="liu"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/a/af/HokkadoU_Member_liu.jpg"><br />
<div class="overlay"><br />
<h3><br />
Yuxiang LIU<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Graduate School of Env.Science<br />
</p><br />
<p><br />
Try something interesting and try something new.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="shehata"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/6/69/IGEM_HokkaidoU_Member_Shehata.jpg"><br />
<div class="overlay"><br />
<h3><br />
Mohamed SHEHATA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Graduate School of Env.Science /<br>Assistant lecturer at Al-Azhar University<br />
</p><br />
<p><br />
I like igem team so much, these students are smart, cooperative, and hard worker, I always learn from them many things.<br />
</p><br />
</div><br />
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<div class="clearfix"></div><br />
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<br />
<div class="member"><br />
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<h2 class="member-title">Special Thanks</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="itoh"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/4/44/HokkaidoU_Member_Itoh_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Takeshi ITOH<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Wiki Advisor<br />
</p><br />
<p><br />
Faculty: Medicine (B4)<br>I was the wiki Jedi in HokkaidoU last year, and now I came back to this team to pass on my experience and help my apprentices to feel the force of HTML! May the force be with you...<br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="kawahata"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/a/a5/HokkaidoU_Member_kawahata_off.png"><br />
<div class="overlay"><br />
<h3><br />
Ami KAWAHATA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Documents Proofreader<br />
</p><br />
<p>Faculty:Science, Biology (B4)<br>Its my third time to participate in iGEM !<br />
Every year I feel the team growing bigger, and better.<br />
iGEM is a great place to change your life, and your world !<br />
have fun if you can</p><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/About_UsTeam:HokkaidoU Japan/About Us2014-10-17T18:29:50Z<p>TaKeZo: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/CSS}}<br />
{{Team:HokkaidoU Japan/About_Us/CSS}}<br />
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<div class="header-title"><br />
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<br />
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<div class="header-title-bottom"><br />
About Us<br />
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<div class="header-picture"><br />
<img src="https://static.igem.org/mediawiki/2014/f/fc/HokkaidoU_Header_kiss.png"><br />
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<span class="menu-list" ><a href="https://2014.igem.org/Team:HokkaidoU_Japan"><span style="position:relative; top:10px;" >Top</span></a></span><!-- Increases to 510px in width--><br />
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<span class="menu-list"><span style="position:relative; top:10px;">RNA in Love</span></span><!-- Increases to 510px in width--><br />
<div class="ldd_submenu" style="left:-415px; width:970px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li><br />
</ul><br />
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<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span><br />
<div class="ldd_submenu" style="width:590px; left:-230px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
</ul><br />
</div><br />
</li><br />
<li class="menu_button"><br />
<span class="menu-list"><span style="position:relative; top:10px;">About Us</span></span><br />
<div class="ldd_submenu" style="width:204px; left: -30px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us">About Us</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
</ul><br />
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<div class="wrapper"><br />
<div id="hokkaidou-contents"><br />
<h1 id="Acknowledgements">Acknowledgements</h1><br />
<h2>Sponsors</h2><br />
<div class="article"><br />
<ul id="sponsor-list"><br />
<li><a href="http://www.aminoup.co.jp/e/"><img src="https://static.igem.org/mediawiki/2014/a/a2/HokkaidoU_sponsorlogo_Aminoup.png"></a></li><br />
<li><a href="http://www.tomoechan.jp/"><img src="https://static.igem.org/mediawiki/2014/f/f2/HokkaidoU_sponsorlogo_Fukuyamaziyouzou.png"></a></li><br />
<li><a href="http://www.hokudai.ac.jp/"><img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_sponsorlogo_Hokudai.png"></a></li><br />
<li><a href="http://www.cosmobio.co.jp/index_e.asp"><img src="https://static.igem.org/mediawiki/2014/f/fa/HokkaidoU_sponsorlogo_Cosmobio.png"></a></li><br />
<li><a href="http://mendel-science.com/"><img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_sponsorlogo_Menderukobo.png"></a></li><br />
<li><a href="http://www.toyobo-global.com/"><img src="https://static.igem.org/mediawiki/2014/b/b5/HokkaidoU_sponsorlogo_Toyobo.png"></a></li><br />
</ul><br />
</div><br />
<br />
<br />
<br />
<h2>Collaborator</h2><br />
<h3>iGEM Japan</h3><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/3/3f/HokkaidoU_Genetic_Learned_Society.jpg"><br />
</div><br />
<p><br />
We attended the 86th Genetic Society of Japan and discuss with 8 Japanese iGEM teams (Gifu, HokkaidoU, Kyoto, Nagahama, Osaka, TMU-tokyo, Tokyo-tech, UT-tokyo).<br />
<br />
<h2>Special thanks</h2><br />
<h3>Nobutaka Nakashima</h3><br />
<h3>Tomohiro Tamura</h3><br />
<br />
<br />
<br />
</p> <br />
<h1 id="Team">Our Team</h1><br />
<div class="fig fig800"><br />
<img id="team-picture" src="https://static.igem.org/mediawiki/2014/4/47/IGEM_HokkaidoU_Japan_Team.jpg" usemap="#Map"><br />
<map name="Map"><br />
<area class="member-click" shape="rect" coords="483,250,527,297 " href="#instructor" ><br />
<area class="member-click" shape="rect" coords="268,189,321,238 " href="#liu" ><br />
<area class="member-click" shape="rect" coords="545,175,594,224" href="#saitoh" ><br />
<area class="member-click" shape="rect" coords="77,95,119,143" href="#kawahata" ><br />
<area class="member-click" shape="rect" coords="232,243,280,289" href="#hana" ><br />
<area class="member-click" shape="rect" coords="310,92,354,148" href="#hana" ><br />
<area class="member-click" shape="rect" coords="368,124,415,174" href="#ayano" ><br />
<area class="member-click" shape="rect" coords="448,105,500,151" href="#osamu" ><br />
<area class="member-click" shape="rect" coords="161,236,204,295" href="#tamaro" ><br />
<area class="member-click" shape="rect" coords="95,236,140,285" href="#tori" ><br />
<area class="member-click" shape="rect" coords="113,187,155,243" href="#tamaro" ><br />
<area class="member-click" shape="rect" coords="312,248,360,290" href="#tori" ><br />
<area class="member-click" shape="rect" coords="342,197,385,246" href="#osamu" ><br />
<area class="member-click" shape="rect" coords="" href="#" ><br />
<area class="member-click" shape="rect" coords="" href="#" ><br />
</map><br />
</div><br />
<br />
<div class="member"><br />
<div class="member-header" id="instructor"><br />
<h2 class="member-title">Instructors</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="ymzk"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2013/4/43/HokkaidoU_2013_Ymzk.png"><br />
<div class="overlay"><br />
<h3><br />
Ken-ici YAMAZAKI<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Group of Environmental Molecular Biology, Section of Environmental Biology, Faculty of Environmental Earth Science<br><br />
<!--(<a href="http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html">Yamazaki Lab</a>)--><br />
</p><br />
</div><br />
</div><br />
</li><br />
<li id="sone"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/0/0e/HokkaidoU_Member_sone.png"><br />
<div class="overlay"><br />
<h3><br />
Teruo SONE<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Group of Applied Mycology, Section of Molecular Biology, Faculty of Agriculture<br><br />
<!--(<a href="http://www.agr.hokudai.ac.jp/oukin/index.html">Applied Mycology Lab</a>)--><br />
</p><br />
</div><br />
</div><br />
</li><br />
<div class="clearfix"></div><br />
</ul><br />
</div><br />
</div><br />
<br />
<br />
<div class="member"><br />
<div class="member-header2" id="undergrads"><br />
<h2 class="member-title">Team Members</h2><br />
</div><br />
<div class="member-list"><br />
<ul><br />
<li id="hana"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/6/60/HokkaidoU_Member_shimoyama_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Hana SHIMOYAMA<br />
</h3><br />
<div class="overlay-content"><br />
<p>Team Leader</p><br />
<p><br />
Faculty: Agriculture Plant physiology (B3)<br><br />
My hobby is cultivating vegetable. <br> I like chili food!! I work in soup-curry shop.<br><br />
I like iGEM,<i> E. coli</i> and team members!!!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="ryotaro"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_Member_nouda_off.JPG"><br />
<div class="overlay"><br />
<h3><br />
Ryotaro NOUDA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Experiment Leader<br />
</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I do what I can do. In other matters, I depend on you!<br><br />
I want to study virology, please teach me it!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="ayano"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/2b/HokkaidoU_Member_hata_off.JPG"><br />
<div class="overlay"><br />
<h3><br />
Ayano HATA<br />
</h3><br />
<div class="overlay-content"><br />
<p>Designer / Charge of Human practice</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I always do what I want to do freely. No one can bother me anyway.<br><br />
I treasure my all experiences because everything I meet makes me new. <br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="naoya"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/3/3b/HokkaidoU_Member_matsumura_on.jpg"><br />
<div class="overlay"><br />
<h3><br />
Naoya MATSUMURA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Modeling Supervisor<br />
</p><br />
<p><br />
Faculty: Medicine (B3)<br><br />
In fact, I love physics better than biology. <br><br />
I hope the mystery of life would be described as an elegant physical equation!<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="osamu"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/df/HokkaidoU_Member_horiguchi_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Osamu HORIGUCHI<br />
</h3><br />
<div class="overlay-content"><br />
<p>wiki Supervisor</p><br />
<p><br />
Faculty: Science Biology (B3)<br><br />
I like reptiles, especially snakes and lizards. <br><br />
If I will be born again, I want to be born as Raptor or something that can fly.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="eri"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/2/26/HokkaidoU_Member_mitobe.JPG"><br />
<div class="overlay"><br />
<h3><br />
Eri MITOBE<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Mathematic Girl<br />
</p><br />
<p><br />
Faculty:Science Mathematics (B3)<br><br />
I love sweets!<br><br />
I want to eat delicious sweets in your country.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="tori"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/1/10/HokkaidoU_Member_touri_on.jpg"><br />
<div class="overlay"><br />
<h3><br />
Tori SUZUKI<br />
</h3><br />
<div class="overlay-contenti"><br />
<p>Part Time Job Soldier</p><br />
<p><br />
Faculty: Fisheries Science,Aquaculture Life Science (B2)<br><br />
I love eating,especially foreign cuisine.<br />
So I'm happy if you tell me your country cuisine.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="mami"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/9/99/HokkaidoU_Member_tanaka.jpg"><br />
<div class="overlay"><br />
<h3><br />
Mami TANAKA<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Primer Designer<br />
</p><br />
<p><br />
Faculty:Fisheries Science, Aquaculture Life Science (B2)<br><br />
I'm in love with planktons, especially Closteriums.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="tamaro"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/d6/HokkaidoU_Member_tamaro_off.jpg"><br />
<div class="overlay"><br />
<h3><br />
Tamaro SAKURAI<br />
</h3><br />
<div class="overlay-content"><br />
<p></p><br />
<p>Faculty:Science Biology (B2)<br>I like sports especially cycle road race and triathlon. <br><br />
I want to study and solve the mechanism that differentiated cell obtains pluripotency.<br />
</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="sanbon"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_Member_sanbon.jpg"><br />
<div class="overlay"><br />
<h3><br />
Masaya MITSUMOTO<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
self-introduction<br />
</p><br />
<p>Faculty:Science Biology (B2)<br><br />
I like funny anime or movies for example, The Hangover, Johnny English, Spongebob SquarePants etc. So if you have some recommendation, please give me the title of them!</p><br />
</div><br />
</div><br />
</li><br />
<br />
<li id="hosoki"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/8/89/HokkaidoU_Member_hosoki.jpg"><br />
<div class="overlay"><br />
<h3><br />
Takuya HOSOKI<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
self-introduction<br />
</p><br />
<p>Faculty:Fisheries Science,Aquaculture Life Science (B2)<br><br />
I love fish.Fish love me.</p><br />
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<br />
<li id="saitoh"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/e/e1/HokkaidoU_Member_saitoh.jpg"><br />
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<h3><br />
Kensuke SAITOH<br />
</h3><br />
<div class="overlay-content"><br />
<p>Faculty:Science, Biology (B4)<br><br />
I enjoy every day very much since I am stimulated by motivated members!</p><br />
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<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/a/af/HokkadoU_Member_liu.jpg"><br />
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<h3><br />
Yuxiang LIU<br />
</h3><br />
<div class="overlay-content"><br />
<p><br />
Graduate School of Env.Science<br />
</p><br />
<p><br />
Try something interesting and try something new.<br />
</p><br />
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<li id="shehata"><br />
<img class="show" class="member-picture" src="https://static.igem.org/mediawiki/2014/6/69/IGEM_HokkaidoU_Member_Shehata.jpg"><br />
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<h3><br />
Mohamed SHEHATA<br />
</h3><br />
<div class="overlay-content"><br />
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Graduate School of Env.Science /<br>Assistant lecturer at Al-Azhar University<br />
</p><br />
<p><br />
I like igem team so much, these students are smart, cooperative, and hard worker, I always learn from them many things.<br />
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Takeshi ITOH<br />
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Wiki Advisor<br />
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Faculty: Medicine (B4)<br>I was the wiki Jedi in HokkaidoU last year, and now I came back to this team to pass on my experience and help my apprentices to feel the force of HTML! Try not... Do, or do not. May the force be with you...<br />
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Ami KAWAHATA<br />
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Documents Proofreader<br />
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<p>Faculty:Science, Biology (B4)<br>Its my third time to participate in iGEM !<br />
Every year I feel the team growing bigger, and better.<br />
iGEM is a great place to change your life, and your world !<br />
have fun if you can</p><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festivalTeam:HokkaidoU Japan/Outreach/Univ festival2014-10-17T18:21:44Z<p>TaKeZo: </p>
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Outreach<br />
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Univ. Festival<br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
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<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
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</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
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<h1>Education</h1><br />
<p>We held a workshop in Hokkaido University Festival, as iGEM HokkaidoU team, following last year. In this event, we tried to explain these things</p><br />
<ul><br />
<li>"What is DNA?" and "DNA is actually familiar to us actualy."</li><br />
<li>“What is iGEM?” and “What is our activity?”</li><br />
</ul><br />
<p>To achieve our goal, we conducted an experiment: “Detection of DNA extraction from banana” and poster sessions: “What is DNA?” , ”What is synthetic biology? ” and “What is iGEM?”</p><br />
<p>In the experiment, participants used only daily materials to get more familiar with DNA.</p><br />
<br />
<h3>How to Exmeriment</h3><br />
<ol><br />
<li>Crush one third of banana with spoon; to break its cell wall</li><br />
<li>Mix well 10% saltine water 30 mL and detergent 2.5 mL; Saltine water makes nucleic <br />
acid and salt. Detergent breaks cell membrane.</li><br />
<li>Pour 1 and 2 into a beaker through coffee filter and filtrate them.</li><br />
<li>After 5minutes or so, pour 100% ethanol 40 mL into the beaker slowly.</li><br />
<li>Then you’ll see some white lump, that’s exactly DNA!!</li><br />
</ol><br />
<br />
<p><br />
This experiment is so easy that people of all ages, from children to the elderly, are able to enjoy it and feel DNA is familiar with them.<br />
</p><br />
<br />
<h3>Poster Session</h3><br />
<br />
<p><br />
In poster session, we made a presentation about four contents; about DNA, gene recombination, iGEM and our project of last year.<br />
</p><br />
<p><br />
We drew posters with pictures and illustrations to let people understand easily and had some conversation with them to know how they understand our explanation. We had a good chance to communicate with people having various background or generation and to practice presentation.<br />
</p><br />
<br />
<h1>Survey</h1><br />
<br />
<p><br />
We took questionnaires from participants of our workshop, 133 people.<br />
</p><br />
<p><br />
We got comments about image for gene recombination, following.<br />
</p><br />
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{{Team:HokkaidoU_Japan/JS}}<br />
{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/LengthTeam:HokkaidoU Japan/Projects/Length2014-10-17T18:10:40Z<p>TaKeZo: </p>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
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<h1>Overview</h1><br />
<br />
<p><br />
It is known that the length of anti-sense sequence is related to its repression efficiency (N. Nakashima <i>et al.</i>, 2006<sup><a href="#cite-1">[1]</a></sup>), but the details of the relation is still unclear. In this study, we made different lengths of anti-sense sequence (Fig. 1). </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/8c/HokkaidoU_length_Overveiw2.png"><br />
<div>Fig. 1 Each anti-sense repress mRNA.</div><br />
</div><br />
<br />
<p><br />
Our experiments on anti-sense RNA (asRNA) sequence and its length reveals the relation. Thus our findings will be a clue for other iGEMers who wants to design their own anti-sense sequence.</p><br />
<br />
<h1>Introduction</h1><br />
<br />
<p><br />
In repressing gene by anti-sense RNA, it is important to determine the length of anti-sense. However, it is difficult to do it. Theoretically, if the length is too long, it doesn’t repress target RNA effectively. The reason is because, the RNA polymerase takes a lot of time to synthesize them, and the diffusion rate of them also gets low. However, too short asRNA also has some problem. </p><br />
<p>The short anti-sense cannot bind to the specific part of mRNA because it has too short complementary sequences of target RNA.</p><br />
<p> In the industrial and academic fields, people hope to use anti-sense that has suitable repression efficiency. For example, you can create knock down recombinant organisms easily by using strong anti-sense, and in iGEM, you can make bio-devices which have a complicated gene network and require fine-tuned gene expression. Gene expression is not only ON or OFF. As stated above, each cases need each repression efficiency. Researchers currently tried to change anti-sense repression efficiency by changing anti-sense’s binding sequence. However, it is found that this method is difficult.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/81/HokkaidoU_length_Gifgifgifff.gif"><br />
<div>Fig. 2 Determining a repression region is defficult.</div><br />
</div><br />
<br />
<p><br />
In this project, we made many kinds of anti-sense which have different lengths. These anti-sense constructs themselves is not useful for you. However, in our method, you can create the anti-sense that you desire. We expect this project will help you decide anti-sense sequences. We'd like to tell scientists and iGEMers how easy and accurate to repress the gene expression by anti-sense RNA.</p><br />
<br />
<br />
<h1 style="font-size:43px;" id="Method"> How to synthesize anti-sense constructs</h1><br />
<p><br />
Insert fragments were synthesized based on BioBrick by PCR. <br />
As Forward primers, "XhoI-P<sub>tet</sub> (-10)" was used for making all fragments. The primer binds to -10 region of P<sub>tet</sub> (BBa_R0040) , and its end has XhoI restriction enzyme site. To change the down stream seqeunce, each reverse primers are designed differently (as90 NcoI, as120 NcoI) (Fig. 3). These primers bind to each specific part of mRFP (BBa_E1010) ,and their ends have NcoI restriction enzyme site. By that way, we can get various length of insert fragments, as90 and as120. As90 is the anti-sense that covers 90 bp of mRNA, and as 120 is the anti-sense that covers 120 bp of mRNA (complement RBS and a part of mRFP sequence.) Of course, the edges of insert fragments have restriction enzymes XhoI, NcoI sites. <br />
<br />
</p><br />
<br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1d/HokkaidoU_length_Fig5.png"><br />
<div>Fig. 3 Synthesizing anti-sense by PCR</div><br />
</div><br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/8/8e/HokkaidoU_length_Method3.png"><br />
<div>Fig. 4 Ligate the insert fragment with H-stem vector</div><br />
</div><br />
<p><br />
<br />
<p><br />
After we finished synthesizing insert fragments, we inserted them into our H-stem vector (anti-sense expression vector) by XhoI and NcoI. <br />
<br />
Then, we measured their repression efficiencies. In the same way, we made as30, as60 on H-stem vector and anti-sense B0034 experiment. We performed repression experiment by using their 4 anti-sense constructs.<br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<h1>How to assay</h1><br />
<p><br />
We performed RT-PCR to confirm the transcription of anti-sense RNA (asRNA) constructs.</p><br />
<br />
<ol><br />
<li>Cultivate the colony in 4 mL LB medium for 16 hours.</li><br />
<li>Centrifuge the 4 mL of culture at 10,000 rpm / for 2 min / at 25&deg;C</li><br />
<li>Remove the supernatant and add M9ZB medium then voltex the pelet.</li><br />
<li>Perform RT-PCR</li><br />
<li>Measure absorbance of 260nm about cDNA.</li><br />
</ol><br />
<br />
<h1 id="Results">Results</h1><br />
<p><br />
Though we measured absorbance of 260nm about cDNA, we could not get any cDNA. After RNA extraction, we confirmed absorbance of 260nm (this is absorbance of nucleic acid). However, after RT-PCR of that prodocuts, we could not confirm the existence of nucleic acid. <br />
<br>Here, we show the discussion.</p><br />
<br />
<p><br />
We estimated there is some problems in RNA extraction. First, we maybe loss RNA during the experiment operation. RNA is degraded easily than DNA because RNase is through the world, soil, air and water, of course in laboratories and human's bodies. We seemed to be careless for about it and overlook RNase's contamination.</p><br />
<br />
<p><br />
Second, Deactivation of DNase seemed incomplete. We used DNase at the end of RNA extraction because extracted sample contain DNA and RNA. To do it, we removed DNA and get only RNA. After all steps of RNA extraction, we deactivate DNase at 65&deg;C for 10 min. Probably, DNase was not deactivated. Because of it, DNase degraded cDNA produced in RT-PCR.</p><br />
<br />
<p><br />
Though we could not be in wiki freeze, we are going to retry. We will perform it being careful in these problems.</p><br />
<br />
<br />
<br />
<br />
<h1 id="Conclusion">Developmental experiment</h1><br />
<br />
<p><br />
We theoretically estimated the repression efficiency of anti-sense is related to the length. Therfore, we can make many kinds of repression efficiency anti-senses by making some length anti-sense. However, to synthesize many kinds of anti-senses, we must prepare each primers. As a future work, we propose an efficient method to synthesize various length of anti-sense.</p><br />
<br />
<h2>Method</h2><br />
<br />
<p><br />
Here, we performed this method by using mRFP expression construct as a target gene.</p><br />
<br />
<h3>Preparation for randomizing</h3><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/2/2c/HokkaidoU_length_Random1.png"><br />
<div>Fig. 5 PrePCR for randomizing</div><br />
</div><br />
<p><br />
<!<br />
<p><br />
Before randomizing, we have to perform some steps to make the effective anti-sense sequences .<br />
First, we performed PCR on mRFP construct. We call this step "prePCR". We used below primers.</p><br />
<br />
<ul style="display:block; padding-left:50px; list-style:none; "><br />
<li>XhoI-P<sub>tet</sub> (-10)</li><br />
<li>mRFP 400dn</li><br />
</ul><br />
<br />
XhoI-P<sub>tet</sub> (-10) is a primer that binds to -10 sequence of P<sub>tet</sub> (BBa_R0040), and its 3 ‘ contains XhoI recognition site that is imperative to ligate with our anti-sense vector (H-stem vector). Because it doesn’t contain -35 sequence, DNA synthesizing starts from P<sub>tet</sub>’s -10 sequence and PCR products don’t contain a functional part as promoter.<br />
<br><br />
mRFP 400dn is a primer that binds to mRFP (BBa_E1010)’s 400 downstream. <br />
<br><br />
PCR products that are amplified by these 2 primes showed in Fig. 5.<br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<br />
<p><br />
Through this step, we can get insert fragments containing SD (Shine-Dargalno) sequence and start codon that are important to effictive repression.</p><br />
<br />
<h3>Randomizing</h3><br />
<p><br />
Next, for DNA synthesizing, we used PCR products synthesized previous step. The recipe showed below.</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/5c/HokkaidoU_length_Random2.png"><br />
<div>Fig. 6 Randomizing recipe</div><br />
</div><br />
<br />
<p><br />
We added Klenow fragment, that is a DNA polymerase functioning on 37C, to the general PCR reaction system using KOD Plus NEO. We call this step "PCR adding Klenow". We used 2 primers.<br />
</p><br />
<br />
<br />
<br />
<br />
<ul style="display:block; padding-left:50px; list-style:none;"><br />
<li>XhoI-P<sub>tet</sub> (-10)</li><br />
<li>NcoI-NNNNNN</li><br />
</ul><br />
<br />
<p><br />
NcoI-NNNNNN has random site containing any nucleotides (A, T, C, G), and these random primers bind to random site of template. Theirs 3 ‘ contains NcoI recognition site that is imperative to ligate with H-stem vector. We explain function of these enzymes and primers in any steps.<br />
<br><br />
In 37C step, Klenow fragment work. It synthesize DNA between XhoI-P<sub>tet</sub> (-10) binding site and NcoI-NNNNNN binding sites that are random. DNA amplified in 3hrs are measurable length. This length is important for next step.</p><br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/f/f5/HokkaidoU_length_Random3.png"><br />
<div>Fig. 7 How to make random anti-sense</div><br />
</div><br />
<p><br />
<br />
<p><br />
Next, KOD Plus NEO starts general PCR system. In this reaction system, XhoI-P<sub>tet</sub> (-10) and DNA fragment amplified by Klenow fragment work as primers. XhoI-P<sub>tet</sub> (-10) bind to specific site of template DNA we hope, but another each primers bind to their specific random site because their sequences are different. Therefore we get some length PCR products.<br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<h3>Detail</h3><br />
<p><br />
We use Klenow fragment for reaction for NcoI-NNNNNN as primer. This primer is only about 6 mer that binds to DNA. Therefore in the reaction of KOD Plus NEO, it cannot anneal DNA because of high temperature. However, to use Klenow fragment and react slowly, DNA synthesizing that use NcoI-NNNNNN as primer becomes possible. By the way, Klenow fragment becomes deactivation through KOD Plus NEO’s reaction system.<br />
<br><br />
We used this DNA fragment as insert. We ligated them with the anti-sense vector, performed transformation in a tube and spread to a plate. Some inserts contain XhoI site and NcoI site at each ends and the other contain NcoI site at both ends (Fig. 4). However, because after through dephosphorylation we ligated them, applied inserts were selected automatically in transformation. </p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/1/1f/HokkaidoU_length_Right_and_wrong.png"><br />
<div>Fig. 8 2 kinds of products are synthesized.</div><br />
</div><br />
<br />
<h2>Results</h2><br />
<br />
<div class="fig fig400" ><br />
<img src="https://static.igem.org/mediawiki/2014/a/a7/HokkaidoU_length_Randomizing_result2.png"><br />
<div>Fig. 9 Results of randomizing</div><br />
</div><br />
<p><br />
<!<br />
<p><br />
We performed an experiment by another recipe. Annealing temperture and PCR cycles differs. Low annealing temperture is better than high temperture because random primers have only 6 base binding region. it is important to synthesize various kinds of fragments that many random primers bind stably. To increase PCR cycles, fragments are amplified. The one side (Recipe A) was annealing temperture 55&deg;C, PCR cycles 20cycles. The other side (Recipe B) was annealing temperture 40&deg;C, PCR cycles 35cycles.<br />
<br>We got some colonies in both plates! More colonies existed in Recipe B than Recipe A. Then, we performed colony PCR about them. However, the result was not desired. <br />
</p><br />
<div class="clearfix"><br />
</div><br />
<br />
<br />
<h2>Discussion</h2><br />
<p><br />
We estimate 2 reasons that Recipe B had more colonies than Recipe A. First reason is, thanks to low annealing temperture, various random primers binded to target gene and amplified insert fragments. Second reason is in more PCR cycles, insert fragments were more amplified.<br />
<br>We performed this experiment in August and we treated the result was failure. Thus we gave up this experiment. However, we found that stem constructs are not PCRed well in the end of September. It is possible that the constructs were complete.</p><br />
<br />
<br><br />
<br><br />
<br />
<ol class="citation-list"><br />
<li id="cite-1">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
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<h1 id="Overview">Overview</h1><br />
<p><br />
The weak point of silencing genes by anti-sense RNA (asRNA) is the instability of RNA. RNA has a shorter half-life than DNA, so it is essential for asRNA to be stabilized to repress mRNA sufficiently. This problem was solved by inserting each-complementary sequeces called stem regions into upstream and downstream of as RNA (N. Nakashima <i>et al.</i>, 2006<sup><a href="#cite-1">[1]</a></sup>). These regions make asRNA form stem-loop structures, which lead to increase the stability of asRNA dramatically.<br />
</p><br />
<br />
<p><br />
We followed in their footsteps. We designed our original stem sequence "H-stem" and produced a BioBricked vector for asRNA expression so that other iGEMers and researchers can easily use stemed asRNA. By using this vector, you can get an asRNA-expressing construct just by inserting PCRed target gene into the vector.<br />
</p><br />
<br />
<br />
<h1 id="asRNA_stabilization">asRNA stabilization by stem structure</h1><br />
<br />
<p><br />
The asRNA region is located between two stem regions.(Fig. 1)<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/HokkaidoU_Hstem_DNA.png"><br />
<div>Fig. 1 stem and anti-sense region on DNA</div><br />
</div><br />
<br />
<p><br />
Transcripted to RNA, stem sequence binds with each other and make the whole RNA form stem-loop structure. (Fig. 2) <br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/f/fd/HokkaidoU_Hstem_RNA.png"><br />
<br />
<div>Fig. 2 stem and anti-sense region on RNA</div><br />
</div><br />
<p><br />
This stem-loop structure contributes to stabilize RNA on the following two points.<br />
</p><br />
<br />
<p><br />
One is the protection from the exonuclease activity of RNase. Since RNase in the cell decomposes single-strand RNA, stemed asRNA is hardly affected by such a degradation.<br />
</p><br />
<br />
<p><br />
The other is an increase of thermodynamical stability by a decrease of Gibbs energy. Stem region produces such a great amount of negative $\Delta G$ that positive $\Delta G$ produced by the curvature of loop region is overwhelmed.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<br />
<br />
<p><br />
In bacterial cells, the decomposability of asRNA to that of target mRNA affects the amount of translated mRNA as shown below. Here, x-axis indicates the transcription level of asRNA to that of mRNA.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/55/HokkaidoU_Hstem_graph.png"><br />
</div><br />
<br />
<p><br />
From that graph, you can see that the stability of asRNA greatly contributes to the repression of mRNA. The detail is discussed below.<br />
</p><br />
<br />
<br />
<h2 id="Modelling">Modelling</h2><br />
<br />
<br />
<meta http-equiv="X-UA-Compatible" CONTENT="IE=EmulateIE7"/><br />
<div><!--モデリングで隠せるのはここから--><br />
<br />
<p><br />
The reaction of asRNA (hereinafter referred to as $X$), mRNA ($Y$) and RNA duplex ($Z$) consisting of the asRNA and the mRNA in a bacterial cell is<br />
</p><br />
<br />
\[ X+Y \overset{k_{\rm bind}}{\underset{k_{\rm unbind}}{\rightleftharpoons}} Z \]<br />
<br />
<p><br />
Besides, mRNA and asRNA are produced and decomposed at all times. So, the ordinary differential equation that represses the whole reaction in the cell is shown below.<br />
</p><br />
<br />
\begin{cases}<br />
\dot{x}=a-bx-k_{\rm bind}xy+k_{\rm unbind}z & \\<br />
\dot{y}=1-y-k_{\rm bind}xy+k_{\rm unbind}z & \\<br />
\dot{z}=k_{\rm bind}xy-k_{\rm unbind}z-cz &<br />
\end{cases}<br />
<br />
<p><br />
Here, we took $1$ for two constants of the right side first and second terms in the second formula using the flexibilities of $[time]$ and $[concentration]$. Thus, constant $a$ stands for the expression level of asRNA to that of target mRNA and constant $b$ the decomposability of asRNA to that of mRNA. <br />
</p><br />
<br />
<p><br />
The unique stable fixed point (in quadrant I) of the ODE is easily found by taking $0$ for each equation. Especially, $y$ value of the fixed point, $y^*$, is<br />
</p><br />
<br />
\[ y=\frac{1}{2} \biggl\{ \sqrt{ \bigl( a-1+\frac{b}{\gamma} \bigl)^2 +4 \frac{b}{\gamma}} - \Bigl( a-1+\frac{b}{\gamma} \Bigl) \biggl\} \]<br />
<br />
<p><br />
Provided that \[ \gamma = \frac{k_{\rm bind}c}{k_{\rm unbind}+c} \]<br />
</p><br />
<br />
<p><br />
We find the graph above by regarding this formula as a function of $a$.<br />
</p><br />
<br />
<p><br />
Though here we let $\gamma$ value be $1$ arbitarily, this value does not affect qualitatively as the formula shows.<br />
</p><br />
</div><!--ここまで!!!!!!--><br />
<br />
<br />
<h1 id="Construct">Construct</h1><br />
<p><br />
<br />
We constructed a vector "H-stem vector" which includes the H-stem system.<br />
<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_Hstem_construct.png"><br />
<div>Fig. 3 The structure of H-stem system</div><br />
</div><br />
<br />
<br />
<p><br />
Stemed asRNA are translated by a inducible promoter, P<sub>lac</sub>, and it is activated by IPTG induction.<br />
</p><br />
<br />
<p><br />
Since anti-sense region can be digested out by NcoI and XhoI, you can insert any anti-sense sequence as recounted above. Additionally, a dummy anti-sense sequence is in advance inserted into this vector.<br />
</p><br />
<br />
<p><br />
<br />
A point to notice when using our H-stem is that, the stem sequence is difficult to PCR, especially in sequencing PCR.<br />
It is supposed that single-strand DNA of this sequence also forms stem-loop structure and it prevents from the extension of complementary strand, but the detailed mechanism is unknown. <br />
Therefore, if you want to sequence the H-stem, you have to divide it's sequence into upstream part and downstream part before doing PCR. <br />
</p><br />
<br />
<h1 id="How_To_Use">How to Use</h1><br />
<br />
<p><br />
Now, we explain how to use H-stem vector.<br />
</p><br />
<br />
<p><br />
What you need is two restriction enzymes, NcoI and XhoI, and two PCR primers, added to the H-stem vector.<br />
</p><br />
<br />
<br><br />
<br />
<ol style="display:block;" type="1"><br />
<li>Determine the asRNA binding sequence, which is about 60 bases, from the tail of RBS to the head of CDS. (Please confirm that the sequence does not include NcoI or XhoI site.)</li><br />
<li>Design the primers which amplify the sequence. Here, add XhoI recognition site (5’-GGG<b>CTCGAG</b>…) to 5’ end of the forward primer and NcoI recognition site (5’-GGG<b>CCATGG</b>…) to that of forward primer.</li><br />
<li>PCR the sequence by the two primers.</li><br />
<li>Digest the PCR product and H-stem vector by NcoI and XhoI, mix them, and ligate together.</li><br />
</ol><br />
<br />
<p><br />
We hope that this method will be a help to many iGEMers.<br />
</p><br />
<br />
<br><br />
<br><br />
<h3>Reference</h3><br />
<ol class="citation-list"><br />
<li id="cite-1">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/Modelling/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_JapanTeam:HokkaidoU Japan2014-10-17T17:43:38Z<p>TaKeZo: </p>
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<h1 style="font-family: 'Special Elite', cursive; font-size: 48px;">~ RNA in love ~</h1><br />
<h3 style="font-family: 'Special Elite', cursive; font-size: 24px;">This year iGEM HokkaidoU invites you to a lovely story ...</h3><br />
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<p><br />
This is a love story of one shy girl called Annie. She was very hesitant, but her passion for him was second to none. One day, Cupid found Annie and helped her. Well, how would the future of her romance going to be?<br />
<br />
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<p style="line-height:10px; font-weight:bold;"><br />
Annie / asRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The shiest girl in the village, but has a feeling to Mike in secrecy.<br />
</p><br />
</div><br />
<br />
<div class="right-up"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/b/b8/HokkaidoU_Top_Book_mRNA.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Mike / mRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most popular boy in the village, but a little indecisive.<br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<div class="page-vertical-bottom"><br />
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<img class="character-pic" src="https://static.igem.org/mediawiki/2014/0/0f/HokkaidoU_Top_Book_Ribosome.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Rachel / ribosome<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most confident girl in the village. She loves Mike and approaches him positively.<br />
</p><br />
</div><br />
<br />
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<img class="character-pic" src="https://static.igem.org/mediawiki/2014/8/8f/HokkaidoU_Top_Book_cupid.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Cupid / HokkaidoU<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
A wonderful sprite in the village. He sometimes helps a villager whimsically.<br />
</p><br />
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</div><br />
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<br />
<br />
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<div style="width:70%; left:50px; top:77px;font-size:23px; line-height:40px;" class="left-page"><br />
Once upon a time, there was a girl, Annie. She fell in love with Mike who was the darling of the town. But she had a formidable rival, Rachel. She was very positive to her love so that she always wanted to monopolize Mike. <br />
</div><br />
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<div style="width:59%; left:350px; top:15px;font-size:23px; line-height:40px;" class="right-page"><br />
Contrasting to her, as Annie was too shy, it was difficult for her only to approach to Mike. Nevertheless, she never stopped thinking about Mike. Cupid found such poor Annie.<br />
</div><br />
</dvi><br />
</div><br />
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<div style="width:70%; right:5%; top:90px;font-size:23px; line-height:40px;" class="right-page"><br />
As Cupid wanted to help Annie’s love, he casted a spell over her. This was the wonderful magic STEM. Enchanted, her clothes turned into greatly beautiful clothes. <br />
</div><br />
</div><br />
<img style="position:absolute; top:225px; left:199px; width:488px; border-radius:50px;" src="https://static.igem.org/mediawiki/2014/3/30/HokkaidoU_Top_Book_kirakira.png"><br />
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<div style="width:65%; left:30px;font-size:23px; line-height:40px; top:41px;" class="left-page"><br />
She was very surprised to look at herself in a mirror because the girl in a mirror appeared like not herself to her. Owing to wonderful magic, she was able to have confidence and a little courage to go to Mike.<br />
</div><br />
<div class="right-page"><br />
<img style="left:633px; bottom:24px; width:362px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/e/e4/HokkaidoU_Top_Book_Transform.png"><br />
</div><br />
<br />
</div><br />
</div><br />
<br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <div style="left:100px; top:89px; width:80%;font-size:23px; line-height:40px;" class="page-vertical-top"><br />
Mike fell in love with changed Annie at a first sight. <br />
“Just a small change of myself makes my world more wonderful than I have dreamed ever.”<br />
Although Annie couldn’t hide her surprise, she felt very happy.<br />
And then, Annie and Mike were together happily forever. <br />
<br />
</div><br />
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</div><!--background--><br />
</html><br />
{{Team:HokkaidoU_Japan/CSS}}<br />
{{Team:HokkaidoU_Japan/Top_Header/CSS}}<br />
{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}<br />
{{Team:HokkaidoU_Japan/JS}}<br />
{{Team:HokkaidoU_Japan/Book/CSS}}<br />
{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_JapanTeam:HokkaidoU Japan2014-10-17T17:42:54Z<p>TaKeZo: </p>
<hr />
<div><html class="demo-1"><br />
<link href='http://fonts.googleapis.com/css?family=Special+Elite' rel='stylesheet' type='text/css'><br />
<link href="//netdna.bootstrapcdn.com/font-awesome/4.0.3/css/font-awesome.css" rel="stylesheet"><br />
<div id="background"><br />
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<div id="top-header"><br />
<div><br />
<img id="header-image" src="https://static.igem.org/mediawiki/2014/7/77/HokkaidoU_Top_Header.png"><br />
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<ul id="ldd_menu" class="ldd_menu"><br />
<li><br />
<span class="menu-list" ><span style="position:relative; top:10px;" href="#">Top</span></span><!-- Increases to 510px in width--><br />
</li><br />
<li class="menu_button"><br />
<span class="menu-list"><span style="position:relative; top:10px;">RNA in Love</span></span><!-- Increases to 510px in width--><br />
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<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li><br />
</ul><br />
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</li><br />
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<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span><br />
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<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us">About Us</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
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<a href="https://igem.org/Main_Page"><img style="position:relative;float:right;top:-40px;right:135px;" src="https://static.igem.org/mediawiki/2014/0/07/HokkaidoU_Top_HQ.png"></a><br />
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<h1 style="font-family: 'Special Elite', cursive; font-size: 48px;">~ RNA in love ~</h1><br />
<h3 style="font-family: 'Special Elite', cursive; font-size: 24px;">This year iGEM HokkaidoU invites you to a lovely story ...</h3><br />
</div><br />
<div id="bb-bookblock" class="bb-bookblock"><br />
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<div class="left-page-top"></div><br />
<div class="right-page-top"><br />
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</div> <br />
</div><br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <br />
<div class="left-page"><br />
<div class="text-box"><br />
<p><br />
This is a love story of one shy girl called Annie. She was very hesitant, but her passion for him was second to none. One day, Cupid found Annie and helped her. Well, how would the future of her romance going to be?<br />
<br />
</p><br />
</div><br />
</div><br />
<div class="right-page"><br />
<div class="page-vertical-top"><br />
<div class="left-up"><br />
<img style="width:101px;margin-bottom:10px;" class="character-pic" src="https://static.igem.org/mediawiki/2014/c/c6/HokkaidoU_Top_Book_as.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Annie / asRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The shiest girl in the village, but has a feeling to Mike in secrecy.<br />
</p><br />
</div><br />
<br />
<div class="right-up"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/b/b8/HokkaidoU_Top_Book_mRNA.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Mike / mRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most popular boy in the village, but a little indecisive.<br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<div class="page-vertical-bottom"><br />
<div class="left-bottom"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/0/0f/HokkaidoU_Top_Book_Ribosome.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Rachel / ribosome<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most confident girl in the village. She loves Mike and approaches him positively.<br />
</p><br />
</div><br />
<br />
<div class="right-bottom"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/8/8f/HokkaidoU_Top_Book_cupid.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Cupid / HokkaidoU<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
A wonderful sprite in the village. He sometimes helps a villager whimsically.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
</div><br />
<br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <br />
<div class="page-vertical-top"><br />
<div style="width:70%; left:50px; top:77px;font-size:23px; line-height:40px;" class="left-page"><br />
Once upon a time, there was a girl, Annie. She fell in love with Mike who was the darling of the town. But she had a formidable rival, Rachel. She was very positive to her love so that she always wanted to monopolize Mike. <br />
</div><br />
<div class="right-page"><br />
<img style="left:745px; width:170px;top:20px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/3/3d/HokkaidoU_Top_Picture_watch.png"><br />
</div><br />
</div><br />
<div class="page-vertical-bottom"><br />
<div class="left-page"><br />
<img style="bottom:12px;left:-50px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/d/d4/HokkaidoU_Top_Picutre_mRNA-Ribosome.png"><br />
</div><br />
<div style="width:59%; left:350px; top:15px;font-size:23px; line-height:40px;" class="right-page"><br />
Contrasting to her, as Annie was too shy, it was difficult for her only to approach to Mike. Nevertheless, she never stopped thinking about Mike. Cupid found such poor Annie.<br />
</div><br />
</dvi><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"><br />
<div class="page-vertical-top"><br />
<div style="width:30%;" class="left-page"><br />
<img style="top:-2px; left:-43px; width:320px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/d/dc/HokkaidoU_Top_Book_Cupid.png"><br />
</div><br />
<div style="width:70%; right:5%; top:90px;font-size:23px; line-height:40px;" class="right-page"><br />
As Cupid wanted to help Annie’s love, he casted a spell over her. This was the wonderful magic STEM. Enchanted, her clothes turned into greatly beautiful clothes. <br />
</div><br />
</div><br />
<img style="position:absolute; top:225px; left:199px; width:488px; border-radius:50px;" src="https://static.igem.org/mediawiki/2014/3/30/HokkaidoU_Top_Book_kirakira.png"><br />
<div class="page-vertical-bottom"><br />
<div style="width:65%; left:30px;font-size:23px; line-height:40px; top:41px;" class="left-page"><br />
She was very surprised to look at herself in a mirror because the girl in a mirror appeared like not herself to her. Owing to wonderful magic, she was able to have confidence and a little courage to go to Mike.<br />
</div><br />
<div class="right-page"><br />
<img style="left:633px; bottom:24px; width:362px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/e/e4/HokkaidoU_Top_Book_Transform.png"><br />
</div><br />
<br />
</div><br />
</div><br />
<br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <div style="left:100px; top:89px; width:80%;font-size:23px; line-height:40px;" class="page-vertical-top"><br />
Mike fell in love with changed Annie at a first sight. <br />
“Just a small change of myself makes my world more wonderful than I have dreamed ever.”<br />
Although Annie couldn’t hide her surprise, she felt very happy.<br />
And then, Annie and Mike were together happily forever. <br />
<br />
</div><br />
<div class="page-vertical-bottom"><br />
<div class="left-page"><br />
<img style="width:113px;bottom:90px;left:-45px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/6/69/HokkaidoU_Top_Book_Ribosome_ki-.png"><br />
<img style="width:180px; bottom:-14px; left:28px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/c/c5/HokkaidoU_Top_Book_one-eye.png"><br />
</div><br />
<div class="right-page"><br />
<img style="width:180px; left:570px; bottom:-28px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/8/81/HokkaidoU_Top_Book_newas.png"><br />
</div><br />
</div><br />
</div><br />
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<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <br />
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<img class="picture-last" src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_Top_Book_last.png"><br />
</div><br />
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<img class="picture-fin" src="https://static.igem.org/mediawiki/2014/8/82/Fin.png"><br />
</div><br />
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</html><br />
{{Team:HokkaidoU_Japan/CSS}}<br />
{{Team:HokkaidoU_Japan/Top_Header/CSS}}<br />
{{Team:HokkaidoU_Japan/LargeDropDown/CSS}}<br />
{{Team:HokkaidoU_Japan/JS}}<br />
{{Team:HokkaidoU_Japan/Book/CSS}}<br />
{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Template:Team:HokkaidoU_Japan/Book/CSSTemplate:Team:HokkaidoU Japan/Book/CSS2014-10-17T17:41:56Z<p>TaKeZo: </p>
<hr />
<div><html><br />
<style type="text/CSS"><br />
<br />
/* General Demo Style */<br />
@import url(http://fonts.googleapis.com/css?family=Lato:300,400,700);<br />
<br />
*, *:after, *:before { -webkit-box-sizing: border-box; -moz-box-sizing: border-box; box-sizing: border-box; }<br />
<br />
/* Clearfix hack by Nicolas Gallagher: http://nicolasgallagher.com/micro-clearfix-hack/ */<br />
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.clearfix:after { clear: both; }<br />
<br />
<br />
/* Header Style */<br />
.main,<br />
.container > header {<br />
margin: 0 auto;<br />
padding: 2em;<br />
}<br />
<br />
.container {<br />
height: 100%;<br />
margin-top:0;<br />
}<br />
<br />
.container > header {<br />
text-align: center;<br />
background: rgba(0,0,0,0.01);<br />
}<br />
<br />
.container > header h1 {<br />
font-size: 2.625em;<br />
line-height: 1.3;<br />
margin: 0;<br />
font-weight: 300;<br />
}<br />
<br />
.container > header span {<br />
display: block;<br />
font-size: 60%;<br />
opacity: 0.3;<br />
padding: 0 0 0.6em 0.1em;<br />
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/* Main Content */<br />
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max-width: 69em;<br />
}<br />
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.column {<br />
float: left;<br />
width: 50%;<br />
padding: 0 2em;<br />
min-height: 300px;<br />
position: relative;<br />
}<br />
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.column:nth-child(2) {<br />
box-shadow: -1px 0 0 rgba(0,0,0,0.1);<br />
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font-weight: 300;<br />
font-size: 2em;<br />
padding: 0;<br />
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}<br />
<br />
/* To Navigation Style */<br />
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background: #fff;<br />
background: rgba(255, 255, 255, 0.6);<br />
text-transform: uppercase;<br />
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.codrops-icon-drop:before {<br />
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<br />
/* Demo Buttons Style */<br />
.codrops-demos {<br />
padding-top: 1em;<br />
font-size: 0.9em;<br />
}<br />
<br />
.codrops-demos a {<br />
display: inline-block;<br />
margin: 0.2em;<br />
padding: 0.45em 1em;<br />
background: #999;<br />
color: #fff;<br />
font-weight: 700;<br />
border-radius: 2px;<br />
}<br />
<br />
.codrops-demos a:hover,<br />
.codrops-demos a.current-demo,<br />
.codrops-demos a.current-demo:hover {<br />
opacity: 0.6;<br />
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<br />
.codrops-nav {<br />
text-align: center;<br />
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.codrops-nav a {<br />
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<br />
.column p {<br />
text-align: left;<br />
font-size: 1.5em;<br />
}<br />
<br />
.column:nth-child(2) {<br />
box-shadow: 0 -1px 0 rgba(0,0,0,0.1);<br />
}<br />
}<br />
<br />
@media screen and (max-width: 25em) {<br />
<br />
.codrops-icon span {<br />
display: none;<br />
}<br />
<br />
}<br />
<br />
.bb-bookblock {<br />
width: 960px;<br />
height: 960px;<br />
margin: 10px auto;<br />
position: relative;<br />
z-index: 100;<br />
-webkit-perspective: 1300px;<br />
-moz-perspective: 1300px;<br />
perspective: 1300px;<br />
-webkit-backface-visibility: hidden;<br />
-moz-backface-visibility: hidden;<br />
backface-visibility: hidden;<br />
}<br />
<br />
.bb-page {<br />
position: absolute;<br />
-webkit-transform-style: preserve-3d;<br />
-moz-transform-style: preserve-3d;<br />
transform-style: preserve-3d;<br />
-webkit-transition-property: -webkit-transform;<br />
-moz-transition-property: -moz-transform;<br />
transition-property: transform;<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-vertical .bb-page {<br />
width: 50%;<br />
height: 100%;<br />
left: 50%;<br />
-webkit-transform-origin: left center;<br />
-moz-transform-origin: left center;<br />
transform-origin: left center;<br />
}<br />
<br />
.bb-horizontal .bb-page {<br />
width: 100%;<br />
height: 50%;<br />
top: 50%;<br />
-webkit-transform-origin: center top;<br />
-moz-transform-origin: center top;<br />
transform-origin: center top;<br />
}<br />
<br />
.bb-page > div,<br />
.bb-outer,<br />
.bb-content,<br />
.bb-inner {<br />
position: absolute;<br />
height: 100%;<br />
width: 100%;<br />
top: 0;<br />
left: 0;<br />
background: #fff;<br />
}<br />
<br />
.bb-outer {<br />
-webkit-backface-visibility: hidden;<br />
-moz-backface-visibility: hidden;<br />
backface-visibility: hidden;<br />
}<br />
<br />
.bb-vertical .bb-content {<br />
width: 200%;<br />
}<br />
<br />
.bb-horizontal .bb-content {<br />
height: 200%;<br />
}<br />
<br />
.bb-page > div {<br />
width: 100%;<br />
-webkit-transform-style: preserve-3d;<br />
-moz-transform-style: preserve-3d;<br />
transform-style: preserve-3d;<br />
}<br />
<br />
.bb-page > div:not(:only-child) {<br />
-webkit-backface-visibility: hidden;<br />
-moz-backface-visibility: hidden;<br />
backface-visibility: hidden;<br />
}<br />
<br />
.bb-vertical .bb-back {<br />
-webkit-transform: rotateY(-180deg);<br />
-moz-transform: rotateY(-180deg);<br />
transform: rotateY(-180deg);<br />
}<br />
<br />
.bb-horizontal .bb-back {<br />
-webkit-transform: rotateX(-180deg);<br />
-moz-transform: rotateX(-180deg);<br />
transform: rotateX(-180deg);<br />
}<br />
<br />
.bb-outer {<br />
width: 100%;<br />
z-index: 999;<br />
overflow:hidden;<br />
}<br />
<br />
.bb-overlay, <br />
.bb-flipoverlay {<br />
background-color: rgba(0, 0, 0, 0.7);<br />
position: absolute;<br />
top: 0px;<br />
left: 0px;<br />
width: 100%;<br />
height: 100%;<br />
opacity: 0;<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-flipoverlay {<br />
background-color: rgba(0, 0, 0, 0.2);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
/* */<br />
<br />
.bb-bookblock.bb-vertical > div.bb-page:first-child,<br />
.bb-bookblock.bb-vertical > div.bb-page:first-child .bb-back {<br />
-webkit-transform: rotateY(180deg);<br />
-moz-transform: rotateY(180deg);<br />
transform: rotateY(180deg);<br />
}<br />
<br />
.bb-bookblock.bb-horizontal > div.bb-page:first-child,<br />
.bb-bookblock.bb-horizontal > div.bb-page:first-child .bb-back {<br />
-webkit-transform: rotateX(180deg);<br />
-moz-transform: rotateX(180deg);<br />
transform: rotateX(180deg);<br />
}<br />
<br />
/* Content display */<br />
.bb-vertical .bb-front .bb-content {<br />
left: -100%;<br />
}<br />
<br />
.bb-horizontal .bb-front .bb-content {<br />
top: -100%;<br />
}<br />
<br />
/* Flipping classes */<br />
.bb-vertical .bb-flip-next,<br />
.bb-vertical .bb-flip-initial {<br />
font-family: 'Special Elite', cursive;<br />
-webkit-transform: rotateY(-180deg);<br />
-moz-transform: rotateY(-180deg);<br />
transform: rotateY(-180deg);<br />
}<br />
<br />
.bb-vertical .bb-flip-prev {<br />
-webkit-transform: rotateY(0deg);<br />
-moz-transform: rotateY(0deg);<br />
transform: rotateY(0deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-horizontal .bb-flip-next,<br />
.bb-horizontal .bb-flip-initial {<br />
-webkit-transform: rotateX(180deg);<br />
-moz-transform: rotateX(180deg);<br />
transform: rotateX(180deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-horizontal .bb-flip-prev {<br />
-webkit-transform: rotateX(0deg);<br />
-moz-transform: rotateX(0deg);<br />
transform: rotateX(0deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-vertical .bb-flip-next-end {<br />
-webkit-transform: rotateY(-15deg);<br />
-moz-transform: rotateY(-15deg);<br />
transform: rotateY(-15deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-vertical .bb-flip-prev-end {<br />
-webkit-transform: rotateY(-165deg);<br />
-moz-transform: rotateY(-165deg);<br />
transform: rotateY(-165deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-horizontal .bb-flip-next-end {<br />
-webkit-transform: rotateX(15deg);<br />
-moz-transform: rotateX(15deg);<br />
transform: rotateX(15deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-horizontal .bb-flip-prev-end {<br />
-webkit-transform: rotateX(165deg);<br />
-moz-transform: rotateX(165deg);<br />
transform: rotateX(165deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-item {<br />
width: 100%;<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
display:none;<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
/* No JS */<br />
.no-js .bb-bookblock, <br />
.no-js ul.bb-custom-grid li {<br />
width: auto;<br />
height: auto;<br />
}<br />
<br />
.no-js .bb-item {<br />
display: block;<br />
position: relative;<br />
}<br />
<br />
<br />
.bb-custom-wrapper {<br />
width: 960px;<br />
position: relative;<br />
margin: 0 auto;<br />
text-align: center;<br />
}<br />
<br />
.bb-custom-wrapper .bb-bookblock {<br />
/*box-shadow: 0 12px 20px -10px rgba(81,64,49,0.6);*/<br />
overflow: hidden;<br />
}<br />
<br />
.bb-custom-wrapper h3 {<br />
font-size: 1.4em;<br />
font-weight: 300;<br />
margin: 0.4em 0 1em;<br />
}<br />
<br />
.bb-custom-wrapper nav {<br />
width: 100%;<br />
height: 30px;<br />
margin: 1em auto 0;<br />
position: relative;<br />
z-index: 0;<br />
text-align: center;<br />
}<br />
<br />
.bb-custom-wrapper nav a {<br />
display: inline-block;<br />
width: 30px;<br />
height: 30px;<br />
text-align: center;<br />
border-radius: 2px;<br />
margin: 2px;<br />
}<br />
<br />
.bb-custom-wrapper nav a:hover {<br />
opacity: 0.6;<br />
}<br />
<br />
<br />
<br />
.left-page-top {<br />
float: left;<br />
width:49%;<br />
height: auto;<br />
position: absolute;<br />
top:50px;<br />
}<br />
<br />
.left-page {<br />
float: left;<br />
width:49%;<br />
height: auto;<br />
position: absolute;<br />
top:0;<br />
}<br />
<br />
.right-page-top {<br />
float: left;<br />
width: 50%;<br />
height: 100%;<br />
position:absolute;<br />
left:50%;<br />
text-align:left;<br />
}<br />
<br />
.right-page {<br />
float: left;<br />
width: 49%;<br />
height: auto;<br />
position:absolute;<br />
top:0;<br />
text-align:left;<br />
}<br />
<br />
.page-title {<br />
margin: 30px auto;<br />
font-size: 40px;<br />
font-weight: bold;<br />
font-family: 'Special Elite', cursive;<br />
position:relative;<br />
top:-600px;<br />
}<br />
<br />
.book-index {<br />
text-align: left;<br />
margin: 20px auto;<br />
list-style: none;<br />
position: absolute;<br />
}<br />
<br />
.book-index > ul > li {<br />
font-size: 25px;<br />
margin: 20px auto;<br />
list-style: none;<br />
}<br />
<br />
.book-index ul {<br />
list-style: none;<br />
line-height:30px;<br />
}<br />
<br />
.book-index > ul > li > ul > li {<br />
font-size: 15px;<br />
list-style: none;<br />
}<br />
<br />
.bb-item-contents {<br />
font-size: 30px;<br />
line-height: 60px;<br />
margin: 25px 100px ;<br />
position: absolute;<br />
}<br />
<br />
.bb-item-picture {<br />
position: relative;<br />
right: -300px;<br />
z-index:101;<br />
}<br />
<br />
.top-picture {<br />
width: 100%;<br />
height: 100%;<br />
}<br />
<br />
.book-background {<br />
width:100%;<br />
height:100%;<br />
position: relative;<br />
}<br />
<br />
.page-vertical-top {<br />
height:300px;<br />
width:100%;<br />
position:absolute;<br />
top:0px;<br />
}<br />
<br />
.page-vertical-bottom {<br />
height:300px;<br />
width:100%;<br />
position:absolute;<br />
top:300px;<br />
}<br />
.page-picture {<br />
width:270px;<br />
position:relative;<br />
}<br />
<br />
.left-up {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 20px;<br />
top:0px;<br />
}<br />
<br />
.right-up {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 10px;<br />
top:0px;<br />
}<br />
<br />
.left-bottom {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 20px;<br />
top:-21px;<br />
}<br />
<br />
.right-bottom {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 10px;<br />
top:-21px;<br />
}<br />
<br />
.character-pic {<br />
width:145px;<br />
position:relative;<br />
left:20px;<br />
top:5px;<br />
}<br />
<br />
.picture-fin {<br />
width:350px;<br />
position:relative;<br />
left:500px;<br />
<br />
}<br />
<br />
.picture-last {<br />
width:70%;<br />
position:relative;<br />
top:150px;<br />
}<br />
<br />
.index-left {<br />
float:left;<br />
width:50%;<br />
position:absolute;<br />
top:60px;<br />
left:100px;<br />
}<br />
<br />
.index-right {<br />
float:left;<br />
width:50%;<br />
position:absolute;<br />
left:530px;<br />
top:60px;<br />
}<br />
<br />
.text-box {<br />
width:75%;<br />
margin:130px auto;<br />
font-size:20px;<br />
}<br />
<br />
.text-box > p {<br />
line-height:45px;<br />
}<br />
<br />
.bb-custom-wrapper nav {<br />
position: absolute;<br />
bottom: 0; left: -100px;<br />
margin: 0;<br />
padding: 0;<br />
width: 1160px;<br />
height: 960px;<br />
z-index: 1000;<br />
}<br />
<br />
#bb-nav-next {<br />
position: absolute;<br />
top: 0; right: 0;<br />
width: 580px;<br />
height: 960px;<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
#bb-nav-prev {<br />
position: absolute;<br />
top: 0; left: 0;<br />
width: 580px;<br />
height: 960px;<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
#bb-nav-next i {<br />
position: absolute;<br />
top: 480px; right: 50px;<br />
margin: -18px -6px 0 0;<br />
padding: 0;<br />
}<br />
<br />
#bb-nav-prev i {<br />
position: absolute;<br />
top: 480px; left: 50px;<br />
margin: -18px 0 0 -6px;<br />
padding: 0;<br />
}<br />
<br />
.bb-page,<br />
.bb-item,<br />
.left-page-top,<br />
.right-page-top,<br />
.bb-flip-prev,<br />
.bb-flip-next,<br />
.bb-flip-prev-end,<br />
.bb-flip-next-end,<br />
.bb-flip-initial,<br />
.bb-front,<br />
.bb-back,<br />
.bb-flipoverlay<br />
{<br />
height: 600px !important;<br />
}<br />
<br />
.bb-item,<br />
.bb-page {<br />
margin: 180px 0;<br />
}<br />
<br />
/* No JS */<br />
.no-js .bb-custom-wrapper {<br />
height: auto;<br />
}<br />
<br />
.no-js .bb-custom-content {<br />
height: 470px;<br />
}<br />
<br />
<br />
</style><br />
</html></div>TaKeZohttp://2014.igem.org/Template:Team:HokkaidoU_Japan/Book/CSSTemplate:Team:HokkaidoU Japan/Book/CSS2014-10-17T17:38:53Z<p>TaKeZo: </p>
<hr />
<div><html><br />
<style type="text/CSS"><br />
<br />
/* General Demo Style */<br />
@import url(http://fonts.googleapis.com/css?family=Lato:300,400,700);<br />
<br />
*, *:after, *:before { -webkit-box-sizing: border-box; -moz-box-sizing: border-box; box-sizing: border-box; }<br />
<br />
/* Clearfix hack by Nicolas Gallagher: http://nicolasgallagher.com/micro-clearfix-hack/ */<br />
.clearfix:before, .clearfix:after { content: ""; display: table; }<br />
.clearfix:after { clear: both; }<br />
<br />
<br />
/* Header Style */<br />
.main,<br />
.container > header {<br />
margin: 0 auto;<br />
padding: 2em;<br />
}<br />
<br />
.container {<br />
height: 100%;<br />
margin-top:0;<br />
}<br />
<br />
.container > header {<br />
text-align: center;<br />
background: rgba(0,0,0,0.01);<br />
}<br />
<br />
.container > header h1 {<br />
font-size: 2.625em;<br />
line-height: 1.3;<br />
margin: 0;<br />
font-weight: 300;<br />
}<br />
<br />
.container > header span {<br />
display: block;<br />
font-size: 60%;<br />
opacity: 0.3;<br />
padding: 0 0 0.6em 0.1em;<br />
}<br />
<br />
/* Main Content */<br />
.main {<br />
max-width: 69em;<br />
}<br />
<br />
.column {<br />
float: left;<br />
width: 50%;<br />
padding: 0 2em;<br />
min-height: 300px;<br />
position: relative;<br />
}<br />
<br />
.column:nth-child(2) {<br />
box-shadow: -1px 0 0 rgba(0,0,0,0.1);<br />
}<br />
<br />
.column p {<br />
font-weight: 300;<br />
font-size: 2em;<br />
padding: 0;<br />
margin: 0;<br />
text-align: right;<br />
line-height: 1.5;<br />
}<br />
<br />
/* To Navigation Style */<br />
.codrops-top {<br />
background: #fff;<br />
background: rgba(255, 255, 255, 0.6);<br />
text-transform: uppercase;<br />
width: 100%;<br />
font-size: 0.69em;<br />
line-height: 2.2;<br />
}<br />
<br />
.codrops-top a {<br />
padding: 0 1em;<br />
letter-spacing: 0.1em;<br />
color: #888;<br />
display: inline-block;<br />
}<br />
<br />
.codrops-top a:hover {<br />
background: rgba(255,255,255,0.95);<br />
color: #333;<br />
}<br />
<br />
.codrops-top span.right {<br />
float: right;<br />
}<br />
<br />
.codrops-top span.right a {<br />
float: left;<br />
display: block;<br />
}<br />
<br />
.codrops-icon:before {<br />
font-family: 'codropsicons';<br />
margin: 0 4px;<br />
speak: none;<br />
font-style: normal;<br />
font-weight: normal;<br />
font-variant: normal;<br />
text-transform: none;<br />
line-height: 1;<br />
-webkit-font-smoothing: antialiased;<br />
}<br />
<br />
.codrops-icon-drop:before {<br />
content: "\e001";<br />
}<br />
<br />
.codrops-icon-prev:before {<br />
content: "\e004";<br />
}<br />
<br />
.codrops-icon-archive:before {<br />
content: "\e002";<br />
}<br />
<br />
.codrops-icon-next:before {<br />
content: "\e000";<br />
}<br />
<br />
.codrops-icon-about:before {<br />
content: "\e003";<br />
}<br />
<br />
/* Demo Buttons Style */<br />
.codrops-demos {<br />
padding-top: 1em;<br />
font-size: 0.9em;<br />
}<br />
<br />
.codrops-demos a {<br />
display: inline-block;<br />
margin: 0.2em;<br />
padding: 0.45em 1em;<br />
background: #999;<br />
color: #fff;<br />
font-weight: 700;<br />
border-radius: 2px;<br />
}<br />
<br />
.codrops-demos a:hover,<br />
.codrops-demos a.current-demo,<br />
.codrops-demos a.current-demo:hover {<br />
opacity: 0.6;<br />
}<br />
<br />
.codrops-nav {<br />
text-align: center;<br />
}<br />
<br />
.codrops-nav a {<br />
display: inline-block;<br />
margin: 20px auto;<br />
padding: 0.3em;<br />
}<br />
<br />
<br />
@media screen and (max-width: 46.0625em) {<br />
.column {<br />
width: 100%;<br />
min-width: auto;<br />
min-height: auto;<br />
padding: 1em; <br />
}<br />
<br />
.column p {<br />
text-align: left;<br />
font-size: 1.5em;<br />
}<br />
<br />
.column:nth-child(2) {<br />
box-shadow: 0 -1px 0 rgba(0,0,0,0.1);<br />
}<br />
}<br />
<br />
@media screen and (max-width: 25em) {<br />
<br />
.codrops-icon span {<br />
display: none;<br />
}<br />
<br />
}<br />
<br />
.bb-bookblock {<br />
width: 960px;<br />
height: 960px;<br />
margin: 10px auto;<br />
position: relative;<br />
z-index: 100;<br />
-webkit-perspective: 1300px;<br />
-moz-perspective: 1300px;<br />
perspective: 1300px;<br />
-webkit-backface-visibility: hidden;<br />
-moz-backface-visibility: hidden;<br />
backface-visibility: hidden;<br />
}<br />
<br />
.bb-page {<br />
position: absolute;<br />
-webkit-transform-style: preserve-3d;<br />
-moz-transform-style: preserve-3d;<br />
transform-style: preserve-3d;<br />
-webkit-transition-property: -webkit-transform;<br />
-moz-transition-property: -moz-transform;<br />
transition-property: transform;<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-vertical .bb-page {<br />
width: 50%;<br />
height: 100%;<br />
left: 50%;<br />
-webkit-transform-origin: left center;<br />
-moz-transform-origin: left center;<br />
transform-origin: left center;<br />
}<br />
<br />
.bb-horizontal .bb-page {<br />
width: 100%;<br />
height: 50%;<br />
top: 50%;<br />
-webkit-transform-origin: center top;<br />
-moz-transform-origin: center top;<br />
transform-origin: center top;<br />
}<br />
<br />
.bb-page > div,<br />
.bb-outer,<br />
.bb-content,<br />
.bb-inner {<br />
position: absolute;<br />
height: 100%;<br />
width: 100%;<br />
top: 0;<br />
left: 0;<br />
background: #fff;<br />
}<br />
<br />
.bb-outer {<br />
-webkit-backface-visibility: hidden;<br />
-moz-backface-visibility: hidden;<br />
backface-visibility: hidden;<br />
}<br />
<br />
.bb-vertical .bb-content {<br />
width: 200%;<br />
}<br />
<br />
.bb-horizontal .bb-content {<br />
height: 200%;<br />
}<br />
<br />
.bb-page > div {<br />
width: 100%;<br />
-webkit-transform-style: preserve-3d;<br />
-moz-transform-style: preserve-3d;<br />
transform-style: preserve-3d;<br />
}<br />
<br />
.bb-page > div:not(:only-child) {<br />
-webkit-backface-visibility: hidden;<br />
-moz-backface-visibility: hidden;<br />
backface-visibility: hidden;<br />
}<br />
<br />
.bb-vertical .bb-back {<br />
-webkit-transform: rotateY(-180deg);<br />
-moz-transform: rotateY(-180deg);<br />
transform: rotateY(-180deg);<br />
}<br />
<br />
.bb-horizontal .bb-back {<br />
-webkit-transform: rotateX(-180deg);<br />
-moz-transform: rotateX(-180deg);<br />
transform: rotateX(-180deg);<br />
}<br />
<br />
.bb-outer {<br />
width: 100%;<br />
z-index: 999;<br />
overflow:hidden;<br />
}<br />
<br />
.bb-overlay, <br />
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</html></div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_JapanTeam:HokkaidoU Japan2014-10-17T17:37:06Z<p>TaKeZo: </p>
<hr />
<div><html class="demo-1"><br />
<link href='http://fonts.googleapis.com/css?family=Special+Elite' rel='stylesheet' type='text/css'><br />
<link href="//netdna.bootstrapcdn.com/font-awesome/4.0.3/css/font-awesome.css" rel="stylesheet"><br />
<div id="background"><br />
<br />
<!-- begin header--><br />
<div id="header-wrapper"><br />
<div id="top-header"><br />
<div><br />
<img id="header-image" src="https://static.igem.org/mediawiki/2014/7/77/HokkaidoU_Top_Header.png"><br />
</div><br />
</div><br />
</div><br />
<!-- end header--><br />
<!-- begin Menu--><br />
<div class="box"><br />
<div id="box-wrapper"> <br />
<a href="http://igemhokkaidou.com"><img style="position:relative; float:left; left:150px;" src="https://static.igem.org/mediawiki/2014/7/75/HokkaidoU_top_logo.png"></a><br />
<ul id="ldd_menu" class="ldd_menu"><br />
<li><br />
<span class="menu-list" ><span style="position:relative; top:10px;" href="#">Top</span></span><!-- Increases to 510px in width--><br />
</li><br />
<li class="menu_button"><br />
<span class="menu-list"><span style="position:relative; top:10px;">RNA in Love</span></span><!-- Increases to 510px in width--><br />
<div class="ldd_submenu" style="left:-415px; width:970px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li><br />
</ul><br />
</div><br />
</li><br />
<li class="menu_button"><br />
<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span><br />
<div class="ldd_submenu" style="width:590px; left:-230px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
</ul><br />
</div><br />
</li><br />
<li class="menu_button"><br />
<span class="menu-list"><span style="position:relative; top:10px;">About Us</span></span><br />
<div class="ldd_submenu" style="width:204px; left: -30px;"><br />
<ul style="margin-left:20px;"><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us">About Us</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
</ul><br />
</div><br />
</li><br />
</ul><br />
<a href="https://igem.org/Main_Page"><img style="position:relative;float:right;top:-40px;right:135px;" src="https://static.igem.org/mediawiki/2014/0/07/HokkaidoU_Top_HQ.png"></a><br />
<br />
</div><br />
</div><br />
<br />
<!--end menu--><br />
<div class="container"><br />
<br />
<div class="bb-custom-wrapper"><br />
<div style="margin: 40px 0 0;"<br />
<h1 style="font-family: 'Special Elite', cursive; font-size: 48px;">~ RNA in love ~</h1><br />
<h3 style="font-family: 'Special Elite', cursive; font-size: 24px;">This year iGEM HokkaidoU invites you to a lovely story ...</h3><br />
</div><br />
<div id="bb-bookblock" class="bb-bookblock"><br />
<div class="bb-item"><br />
<div class="left-page-top"></div><br />
<div class="right-page-top"><br />
<img class="top-picture" src="https://static.igem.org/mediawiki/2014/9/90/Cover1.png"> <br />
</div> <br />
</div><br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <br />
<div class="left-page"><br />
<div class="text-box"><br />
<p><br />
This is a love story of one shy girl called Annie. She was very hesitant, but her passion for him was second to none. One day, Cupid found Annie and helped her. Well, how would the future of her romance going to be?<br />
<br />
</p><br />
</div><br />
</div><br />
<div class="right-page"><br />
<div class="page-vertical-top"><br />
<div class="left-up"><br />
<img style="width:101px;margin-bottom:10px;" class="character-pic" src="https://static.igem.org/mediawiki/2014/c/c6/HokkaidoU_Top_Book_as.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Annie / asRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The shiest girl in the village, but has a feeling to Mike in secrecy.<br />
</p><br />
</div><br />
<br />
<div class="right-up"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/b/b8/HokkaidoU_Top_Book_mRNA.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Mike / mRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most popular boy in the village, but a little indecisive.<br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<div class="page-vertical-bottom"><br />
<div class="left-bottom"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/0/0f/HokkaidoU_Top_Book_Ribosome.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Rachel / ribosome<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most confident girl in the village. She loves Mike and approaches him positively.<br />
</p><br />
</div><br />
<br />
<div class="right-bottom"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/8/8f/HokkaidoU_Top_Book_cupid.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Cupid / HokkaidoU<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
A wonderful sprite in the village. He sometimes helps a villager whimsically.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
</div><br />
<br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <br />
<div class="page-vertical-top"><br />
<div style="width:70%; left:50px; top:77px;font-size:23px; line-height:40px;" class="left-page"><br />
Once upon a time, there was a girl, Annie. She fell in love with Mike who was the darling of the town. But she had a formidable rival, Rachel. She was very positive to her love so that she always wanted to monopolize Mike. <br />
</div><br />
<div class="right-page"><br />
<img style="left:745px; width:170px;top:20px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/3/3d/HokkaidoU_Top_Picture_watch.png"><br />
</div><br />
</div><br />
<div class="page-vertical-bottom"><br />
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Contrasting to her, as Annie was too shy, it was difficult for her only to approach to Mike. Nevertheless, she never stopped thinking about Mike. Cupid found such poor Annie.<br />
</div><br />
</dvi><br />
</div><br />
</div><br />
<br />
<br />
<br />
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</div><br />
<div style="width:70%; right:5%; top:90px;font-size:23px; line-height:40px;" class="right-page"><br />
As Cupid wanted to help Annie’s love, he casted a spell over her. This was the wonderful magic STEM. Enchanted, her clothes turned into greatly beautiful clothes. <br />
</div><br />
</div><br />
<img style="position:absolute; top:225px; left:199px; width:488px; border-radius:50px;" src="https://static.igem.org/mediawiki/2014/3/30/HokkaidoU_Top_Book_kirakira.png"><br />
<div class="page-vertical-bottom"><br />
<div style="width:65%; left:30px;font-size:23px; line-height:40px; top:41px;" class="left-page"><br />
She was very surprised to look at herself in a mirror because the girl in a mirror appeared like not herself to her. Owing to wonderful magic, she was able to have confidence and a little courage to go to Mike.<br />
</div><br />
<div class="right-page"><br />
<img style="left:633px; bottom:24px; width:362px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/e/e4/HokkaidoU_Top_Book_Transform.png"><br />
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<br />
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<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"> <div style="left:100px; top:89px; width:80%;font-size:23px; line-height:40px;" class="page-vertical-top"><br />
Mike fell in love with changed Annie at a first sight. <br />
“Just a small change of myself makes my world more wonderful than I have dreamed ever.”<br />
Although Annie couldn’t hide her surprise, she felt very happy.<br />
And then, Annie and Mike were together happily forever. <br />
<br />
</div><br />
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{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_JapanTeam:HokkaidoU Japan2014-10-17T17:36:04Z<p>TaKeZo: </p>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
</ul><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
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<span class="menu-list"><span style="position:relative; top:10px;">Outreach</span></span><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival">Univ. Festival</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Univ_festival#Survey">Survey</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey">High-School</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Education">Education</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Survey#Survey">Survey</a></li><br />
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion">Discussion</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
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<h1>~ RNA in love ~</h1><br />
<h3>This year iGEM HokkaidoU invites you to a lovely story ...</h3><br />
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<p><br />
This is a love story of one shy girl called Annie. She was very hesitant, but her passion for him was second to none. One day, Cupid found Annie and helped her. Well, how would the future of her romance going to be?<br />
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<p style="line-height:10px; font-weight:bold;"><br />
Annie / asRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The shiest girl in the village, but has a feeling to Mike in secrecy.<br />
</p><br />
</div><br />
<br />
<div class="right-up"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/b/b8/HokkaidoU_Top_Book_mRNA.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Mike / mRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most popular boy in the village, but a little indecisive.<br />
</p><br />
</div><br />
<br />
</div><br />
<br />
<div class="page-vertical-bottom"><br />
<div class="left-bottom"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/0/0f/HokkaidoU_Top_Book_Ribosome.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Rachel / ribosome<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The most confident girl in the village. She loves Mike and approaches him positively.<br />
</p><br />
</div><br />
<br />
<div class="right-bottom"><br />
<img class="character-pic" src="https://static.igem.org/mediawiki/2014/8/8f/HokkaidoU_Top_Book_cupid.png"><br />
<p style="line-height:10px; font-weight:bold;"><br />
Cupid / HokkaidoU<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
A wonderful sprite in the village. He sometimes helps a villager whimsically.<br />
</p><br />
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</div><br />
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<br />
<br />
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<div style="width:70%; left:50px; top:77px;font-size:23px; line-height:40px;" class="left-page"><br />
Once upon a time, there was a girl, Annie. She fell in love with Mike who was the darling of the town. But she had a formidable rival, Rachel. She was very positive to her love so that she always wanted to monopolize Mike. <br />
</div><br />
<div class="right-page"><br />
<img style="left:745px; width:170px;top:20px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/3/3d/HokkaidoU_Top_Picture_watch.png"><br />
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</div><br />
<div style="width:59%; left:350px; top:15px;font-size:23px; line-height:40px;" class="right-page"><br />
Contrasting to her, as Annie was too shy, it was difficult for her only to approach to Mike. Nevertheless, she never stopped thinking about Mike. Cupid found such poor Annie.<br />
</div><br />
</dvi><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div class="bb-item"><br />
<img class="book-background" src="https://static.igem.org/mediawiki/2014/3/31/HokkaidoU_Top_Book_Background.png"><br />
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<img style="top:-2px; left:-43px; width:320px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/d/dc/HokkaidoU_Top_Book_Cupid.png"><br />
</div><br />
<div style="width:70%; right:5%; top:90px;font-size:23px; line-height:40px;" class="right-page"><br />
As Cupid wanted to help Annie’s love, he casted a spell over her. This was the wonderful magic STEM. Enchanted, her clothes turned into greatly beautiful clothes. <br />
</div><br />
</div><br />
<img style="position:absolute; top:225px; left:199px; width:488px; border-radius:50px;" src="https://static.igem.org/mediawiki/2014/3/30/HokkaidoU_Top_Book_kirakira.png"><br />
<div class="page-vertical-bottom"><br />
<div style="width:65%; left:30px;font-size:23px; line-height:40px; top:41px;" class="left-page"><br />
She was very surprised to look at herself in a mirror because the girl in a mirror appeared like not herself to her. Owing to wonderful magic, she was able to have confidence and a little courage to go to Mike.<br />
</div><br />
<div class="right-page"><br />
<img style="left:633px; bottom:24px; width:362px;" class="page-picture" src="https://static.igem.org/mediawiki/2014/e/e4/HokkaidoU_Top_Book_Transform.png"><br />
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Mike fell in love with changed Annie at a first sight. <br />
“Just a small change of myself makes my world more wonderful than I have dreamed ever.”<br />
Although Annie couldn’t hide her surprise, she felt very happy.<br />
And then, Annie and Mike were together happily forever. <br />
<br />
</div><br />
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<div class="left-page"><br />
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{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_JapanTeam:HokkaidoU Japan2014-10-17T17:33:03Z<p>TaKeZo: </p>
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<h1>~ RNA in love ~</h1><br />
<h3>This year iGEM HokkaidoU invites you to a lovely story ...</h3><br />
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<p><br />
This is a love story of one shy girl called Annie. She was very hesitant, but her passion for him was second to none. One day, Cupid found Annie and helped her. Well, how would the future of her romance going to be?<br />
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<p style="line-height:10px; font-weight:bold;"><br />
Annie / asRNA<br />
</p><br />
<p style="line-height:20px; font-size:15px;"><br />
The shiest girl in the village, but has a feeling to Mike in secrecy.<br />
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Mike / mRNA<br />
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The most popular boy in the village, but a little indecisive.<br />
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Rachel / ribosome<br />
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The most confident girl in the village. She loves Mike and approaches him positively.<br />
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Cupid / HokkaidoU<br />
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<p style="line-height:20px; font-size:15px;"><br />
A wonderful sprite in the village. He sometimes helps a villager whimsically.<br />
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Once upon a time, there was a girl, Annie. She fell in love with Mike who was the darling of the town. But she had a formidable rival, Rachel. She was very positive to her love so that she always wanted to monopolize Mike. <br />
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Contrasting to her, as Annie was too shy, it was difficult for her only to approach to Mike. Nevertheless, she never stopped thinking about Mike. Cupid found such poor Annie.<br />
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</dvi><br />
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As Cupid wanted to help Annie’s love, he casted a spell over her. This was the wonderful magic STEM. Enchanted, her clothes turned into greatly beautiful clothes. <br />
</div><br />
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She was very surprised to look at herself in a mirror because the girl in a mirror appeared like not herself to her. Owing to wonderful magic, she was able to have confidence and a little courage to go to Mike.<br />
</div><br />
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Mike fell in love with changed Annie at a first sight. <br />
“Just a small change of myself makes my world more wonderful than I have dreamed ever.”<br />
Although Annie couldn’t hide her surprise, she felt very happy.<br />
And then, Annie and Mike were together happily forever. <br />
<br />
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{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_JapanTeam:HokkaidoU Japan2014-10-17T17:31:33Z<p>TaKeZo: </p>
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~RNA in LOVE~<br />
This year iGEM HokkaidoU invites you to a lovely story ...<br />
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This is a love story of one shy girl called Annie. She was very hesitant, but her passion for him was second to none. One day, Cupid found Annie and helped her. Well, how would the future of her romance going to be?<br />
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Annie / asRNA<br />
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The shiest girl in the village, but has a feeling to Mike in secrecy.<br />
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Mike / mRNA<br />
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The most popular boy in the village, but a little indecisive.<br />
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Rachel / ribosome<br />
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The most confident girl in the village. She loves Mike and approaches him positively.<br />
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Cupid / HokkaidoU<br />
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A wonderful sprite in the village. He sometimes helps a villager whimsically.<br />
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Once upon a time, there was a girl, Annie. She fell in love with Mike who was the darling of the town. But she had a formidable rival, Rachel. She was very positive to her love so that she always wanted to monopolize Mike. <br />
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Contrasting to her, as Annie was too shy, it was difficult for her only to approach to Mike. Nevertheless, she never stopped thinking about Mike. Cupid found such poor Annie.<br />
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As Cupid wanted to help Annie’s love, he casted a spell over her. This was the wonderful magic STEM. Enchanted, her clothes turned into greatly beautiful clothes. <br />
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She was very surprised to look at herself in a mirror because the girl in a mirror appeared like not herself to her. Owing to wonderful magic, she was able to have confidence and a little courage to go to Mike.<br />
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Mike fell in love with changed Annie at a first sight. <br />
“Just a small change of myself makes my world more wonderful than I have dreamed ever.”<br />
Although Annie couldn’t hide her surprise, she felt very happy.<br />
And then, Annie and Mike were together happily forever. <br />
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{{Team:HokkaidoU_Japan/Footer/CSS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034Team:HokkaidoU Japan/Projects/asB00342014-10-17T17:25:43Z<p>TaKeZo: </p>
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Anti-Sense B0034<br />
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<h1>Overview</h1><br />
<br />
<p>Anti-sense RNA is being studied actively over the world. However, reliable method for silencing genes has not been clarified. It is hard to find efficient sequence of asRNAs and to synthesize new anti-sense fragments matching your target genes. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/8/88/HokkaidoU_project_antisenseB0034_overview01_800.png"><br />
<div>Fig. 1 You have to modify asRNAs conforming with each target gene.</div><br />
</div><br />
<p></p><p></p><br />
<br />
<p>We thought it is useful to use a common anti-sense sequence for various target genes.<br />
</p><br />
<br />
<p></p><p></p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/3/38/HokkaidoU_project_antisenseB0034_overview02_800.png"><br />
<div>Fig. 2 Concept of common anti-sence sequence.</div><br />
</div><br />
<br />
<p></p><p></p><br />
<p>We found that anti-sense RBS (B0034) fragment, which is used by some iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.<br />
</p><br />
<p></p><br />
<p><br />
We also synthesized anti-sense B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.<br />
</p><br />
<p><br />
Specific anti-sense RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. All you have to do is to add our anti-sense fragment to the construct with the target gene!!<br />
</p><br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/2/28/HokkaidoU_project_antisenseB0034_overview03_800.png"><br />
<div>Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS</div><br />
</div><br />
<br />
<h1 style="font-size:43px;" id="Method">How to synthesize anti-sense constructs</h1><br />
<p>Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI. </p><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d1/HokkaidoU_project_antisenseB0034_method02_400.png"><br />
<div>Fig. 1 How to make anti-sense B0034 by primer annealing</div><br />
</div><br />
<br />
<div class="fig fig400 para"><p><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_project_antisenseB0034_method03_400.png"><br />
<div>Fig. 2 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense conplex. </div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/0/05/HokkaidoU_antisenseB0034_overview11.png"><br />
<div>Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site</div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cb/HokkaidoU_project_antisenseB0034_method01_400.png"><br />
<div>Fig. 4 B0034 & B0032 sequence </div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/3/37/HokkaidoU_project_antisenseB0034_method06_400.png"><br />
<div>Fig. 5 Our parts</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<br />
<h1><p>How to assay</h1><br />
<p>We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 20 hours</li><br />
<li>To control turbidity up to 0.1 at OD<sub>600</sub></li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 9 hour</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/59/HokkaidoU_project_antisenseB0034_method04_800.png"><br />
<div>Fig. 6 Anti-sense B0034 is induced by IPTG</div><br />
</div><br />
<br />
<h1 id="Results">Preliminary results</h1> <br />
<p><br />
We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034 </li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 2 mL LB culture for about 18 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 18 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cd/HokkaidoU_project_antisenseB0034_result01.png"><br />
<div>Fig. 1 Value of fluorescence of IPTG with and without each sample</div><br />
</div><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/7/76/HokkaidoU_project_antisenseB0034_result02.png"><br />
<div>Fig. 2 Rate of IPTG(+) and IPTG(-)<br />
Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression. </div><br />
</div><br />
<br />
<p>As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.</p><br />
<br />
<h1>Result</h1><br />
<p>We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem.<br />
All antisenses are on pSB1A3 and all target genes are on pSB4C5.<br />
Samples are following</p><br />
<h3>Our samples are following four.</h3><br />
<ul><br />
<li>target B0034+ antisense B0034</li><br />
<li>target B0034+ antisense B0032 </li><br />
<li>target B0032+ antisense B0034 </li><br />
<li>target B0032+ antisense B0032 </li><br />
</ul><br />
<p>We examined each samples with And without IPTG induction.</p><br />
<br />
<h3><p>The way of assay is following.</h3><br />
We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.</p><br />
<br />
<ol><br />
<li>To cultivate the colony in 4 mL LB culture for about 22 hours</li><br />
<li>To control turbidity up to 0.1 at OD600</li><br />
<li>To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)</li><br />
<li>To measure fluorescence after 30 hours</li><br />
</ol><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/4/4d/HokkaidoU_project_antisenseB0034_result03.png"><br />
<div>Fig. 3 Value of fluorescence of IPTG with and without each sample </div><br />
</div><br />
<br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/2/27/HokkaidoU_project_antisenseB0034_result04.png"><br />
<div>Fig. 4 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.</div><br />
</div><br />
<div class="fig fig400 para"><br />
<img src="https://static.igem.org/mediawiki/2014/4/43/HokkaidoU_project_antisenseB0034_result05.png"><br />
<div>Fig. 5 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.</div><br />
</div><br />
<div class="clearfix"></div><br />
<br />
<p><br />
From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.</p><br />
<br />
<h2>Discussion</h2><br />
<ol><br />
<li>Anti-sense was not induced by IPTG; it is leaking.</li><br />
Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.</p><br />
<br />
<p><br />
<li>Antisense did not show any expression</li><br />
Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express.<br />
We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.<br />
</p><br />
<p><br />
<li>Instability of copy number of target gene</li><br />
Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite.<br />
Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.<br />
</p><br />
</ol><br />
<br />
<br />
<h1>Improvement</h1><br />
<p><br />
<ol><br />
<li>Analysis by RT-PCR</li><br />
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG.<br />
We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.<br />
</p><br />
<p><br />
<li>Improvement of medium to induce anti-sense by adding IPTG easily</li><br />
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.<br />
</p><br />
<p><br />
<li>Stability of copy number in target gene</li><br />
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.<br />
</p><br />
<p>We are going to show positive result in Boston!</p><br />
<br />
<br />
<h1 id="Conclusion">Conclusion</h1><br />
<p>We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.</p><br />
<br />
<h3>Instability of mRFP expression</h3><br />
<p>It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/d/d2/HokkaidoU_project_antisenseB0034_overview04.png"><br />
<div>Fig. 6 Rate of RBS used BioBrick parts</div><br />
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<h1>Background</h1><br />
<p><br />
Discovering of non-coding RNA (ncRNAs) and reverse transcription of virus composes the central dogma that was declared by Francis Click. <br />
Since ncRNAs do not encode any proteins, they had been thought that they don't have any functions.<br />
However, it was proved that they do have many functions.<br />
For example, some ncRNAs reduce the efficiency of translation, while others promote that. (C. Claudia <i>et al.</i>, 2012<sup><a href="#cite-1">[1]</a></sup>). <br />
</p> <br />
<p><br />
There are ncRNAs called anti-sense RNA (asRNAs), which down-regulate translation. An asRNA has a complement sequence of its target mRNA and reduces the translation efficiency by hybridizing. <br />
asRNAs can be easily synthesized, but there is no clear method to make stable, highly efficient asRNAs. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/9/93/HokkaidoU_Background01.png"><br />
<div><br />
Fig. 1 An asRNA hybridizes with target mRNA and interrupts entry of ribosome.<br />
</div><br />
</div><br />
<br />
<br />
<div class="fig fig400"><br />
<img style="width:300px;" src="https://static.igem.org/mediawiki/2014/f/fd/HokkaidoU_Hstem_RNA.png"><br />
<div><br />
Fig. 2 Stem structure stabilizes asRNAs<br />
</div><br />
</div><br />
<br />
<p><br />
In this situation, Nakashima and his colleagues discovered asRNA that is sandwiched by 38 base pairs of repeated sequence is stable and regulates translation at a high efficiency (N. Nakashima <i>et al.</i>, 2006<sup><a href="#cite-2">[2]</a></sup>).<br />
</p><br />
<p><br />
Many iGEM teams prepare their project using asRNA, but how many teams use the asRNA that is designed on clear concepts?<br />
This year, we provide you a BioBrick standardized procedure to design asRNAs.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<p><br />
Our procedure to design asRNAs will assure you that<br />
</p><br />
<ul><br />
<li>The asRNAs are based on a clear design concept.</li><br />
<li>They are calculated to have ideal second structures.</li><br />
<li>They have high efficiency to regulate translation, based on our results. </li><br />
</ul><br />
<p><br />
Using this procedure, we arranged these projects.<br />
</p><br />
<ul><br />
<li>Declared a standard procedure to design asRNAs that has modified stem sequence.</li><br />
<li>Synthesized B0034 - most popular RBS - specific asRNA, and widely regulated translation efficiency.</li><br />
<li>Synthesized varios length asRNAs and determined the optimum length. </li><br />
</ul><br />
<p><br />
Our project provides not only highly efficient asRNAs but also brand new viewpoints to your project.<br />
As yet, almost all the projects presented to iGEM have focused on to regulate the behavior of <i>E. coli</i>, only by transforming plasmids.<br />
But now, you can use our asRNAs and regulate expression of the genes which are on genome.<br />
</p><br />
<p><br />
Hybridization of a mRNA and a specific asRNA seems like a love story in the RNA world, so we named our project "RNA in love".<br />
Let's watch over RNA in love!<br />
</p><br />
<br />
<br />
<br />
<br><br />
<br><br />
<ol class="citation-list"><br />
<li id="cite-1">C Claudia<i>et al.</i> (2012) Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491: 7424 454-</li><br />
<li id="cite-2">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/BackgroundTeam:HokkaidoU Japan/Projects/Overview/Background2014-10-17T16:53:16Z<p>TaKeZo: </p>
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li><br />
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<h1>Background</h1><br />
<p><br />
Discovering of non-coding RNA (ncRNAs) and reverse transcription of virus composes the central dogma that was declared by Francis Click. <br />
Since ncRNAs do not encode any proteins, they had been thought that they don't have any functions.<br />
However, it was proved that they do have many functions.<br />
For example, some ncRNAs reduce the efficiency of translation, while others promote that. (C. Claudia <i>et al.</i>, 2012<sup><a href="#cite-1">[1]</a></sup>). <br />
</p> <br />
<p><br />
There are ncRNAs called anti-sense RNA (asRNAs), which down-regulate translation. An asRNA has a complement sequence of its target mRNA and reduces the translation efficiency by hybridizing. <br />
asRNAs can be easily synthesized, but there is no clear method to make stable, highly efficient asRNAs. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/9/93/HokkaidoU_Background01.png"><br />
<div><br />
Fig. 1 An asRNA hybridizes with target mRNA and interrupts entry of ribosome.<br />
</div><br />
</div><br />
<br />
<br />
<div class="fig fig400"><br />
<img style="width:300px;" src="https://static.igem.org/mediawiki/2014/f/fd/HokkaidoU_Hstem_RNA.png"><br />
<div><br />
Fig. 2 Stem structure stabilizes asRNAs<br />
</div><br />
</div><br />
<br />
<p><br />
In this situation, Nakashima and his colleagues discovered asRNA that is sandwiched by 38 base pairs of repeated sequence is stable and regulates translation at a high efficiency (N Nakashima <i>et al.</i>, 2006<sup><a href="#cite-2">[2]</a></sup>).<br />
</p><br />
<p><br />
Many iGEM teams prepare their project using asRNA, but how many teams use the asRNA that is designed on clear concepts?<br />
This year, we provide you a BioBrick standardized procedure to design asRNAs.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<p><br />
Our procedure to design asRNAs will assure you that<br />
</p><br />
<ul><br />
<li>The asRNAs are based on a clear design concept.</li><br />
<li>They are calculated to have ideal second structures.</li><br />
<li>They have high efficiency to regulate translation, based on our results. </li><br />
</ul><br />
<p><br />
Using this procedure, we arranged these projects.<br />
</p><br />
<ul><br />
<li>Declared a standard procedure to design asRNAs that has modified stem sequence.</li><br />
<li>Synthesized B0034 - most popular RBS - specific asRNA, and widely regulated translation efficiency.</li><br />
<li>Synthesized varios length asRNAs and determined the optimum length. </li><br />
</ul><br />
<p><br />
Our project provides not only highly efficient asRNAs but also brand new viewpoints to your project.<br />
As yet, almost all the projects presented to iGEM have focused on to regulate the behavior of <i>E. coli</i>, only by transforming plasmids.<br />
But now, you can use our asRNAs and regulate expression of the genes which are on genome.<br />
</p><br />
<p><br />
Hybridization of a mRNA and a specific asRNA seems like a love story in the RNA world, so we named our project "RNA in love".<br />
Let's watch over RNA in love!<br />
</p><br />
<br />
<br />
<br />
<br><br />
<br><br />
<ol class="citation-list"><br />
<li id="cite-1">C Claudia<i>et al.</i> (2012) Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491: 7424 454-</li><br />
<li id="cite-2">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/BackgroundTeam:HokkaidoU Japan/Projects/Overview/Background2014-10-17T16:44:19Z<p>TaKeZo: </p>
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<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Background">Background</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/Achievements">Achievements</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem">H-stem System</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Overview">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#asRNA_stabilization">Stabilization</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Modelling">Modelling</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#Construct">Construct</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/H_Stem#How_To_Use">How To Use</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Anti-sense B0034</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/asB0034#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length">Length Variation</a></li><br />
<li class="ldd_contents"><a href="">Overview</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Method">Method</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Results">Results</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Projects/Length#Conclusion">Conclusion</a></li><br />
</ul><br />
<ul><br />
<li class="ldd_heading"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Materials">Extra Materials</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Parts">Parts</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Notebook">Notebook</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Safety">Safety</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/Outreach/Discussion#Estimation">Evaluation</a></li><br />
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<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Acknowledgements">Acknowledgements</a></li><br />
<li class="ldd_contents"><a href="https://2014.igem.org/Team:HokkaidoU_Japan/About_Us#Team">Team</a></li><br />
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<div id="hokkaidou-contents"><br />
<h1>Background</h1><br />
<p><br />
Discovering of non-coding RNA (ncRNAs) and reverse transcription of virus composes the central dogma that was declared by Francis Click. <br />
Since ncRNAs do not encode any proteins, they had been thought that they don't have any functions.<br />
However, it was proved that they do have many functions.<br />
For example, some ncRNAs reduce the efficiency of translation, while others promote that. (C. Claudia <i>et al.</i>, 2012<sup><a href="#cite-1">[1]</a></sup>). <br />
</p> <br />
<p><br />
There are ncRNAs called anti-sense RNA (asRNAs), which down-regulate translation. An asRNA has a complement sequence of its target mRNA and reduces the translation efficiency by hybridizing. <br />
asRNAs can be easily synthesized, but there is no clear method to make stable, highly efficient asRNAs. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/9/93/HokkaidoU_Background01.png"><br />
<div><br />
Fig. 1 An asRNA hybridizes with target mRNA and interrupts entry of ribosome.<br />
</div><br />
</div><br />
<br />
<br />
<div class="fig fig400"><br />
<img style="width:300px;" src="https://static.igem.org/mediawiki/2014/f/fd/HokkaidoU_Hstem_RNA.png"><br />
<div><br />
Fig. 2 Stem structure stabilizes asRNAs<br />
</div><br />
</div><br />
<br />
<p><br />
In this situation, Nakashima and his colleagues discovered asRNA that is sandwiched by 38 base pairs of repeated sequence is stable and regulates translation at a high efficiency (N Nakashima <i>et al.</i>, 2006<sup><a href="#cite-2">[2]</a></sup>).<br />
</p><br />
<p><br />
Many iGEM teams prepare their project using asRNA, but how many teams use the asRNA that is designed on clear concepts?<br />
This year, we provide you a BioBrick standardized procedure to design asRNAs.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<p><br />
Our procedure to design asRNAs will assure you that<br />
</p><br />
<ul><br />
<li>The asRNAs are based on a clear design concept.</li><br />
<li>They are calculated to have ideal second structures.</li><br />
<li>They have high efficiency to regulate translation, based on our results. </li><br />
</ul><br />
<p><br />
By using those asRNAs, we did the projects below.<br />
</p><br />
<ul><br />
<li>Construct our asRNA that has modified stem sequence.</li><br />
<li> Construct B0034 -most popular RBS- specific asRNA, and regulate translation efficiency generally.</li><br />
<li> Construct various length asRNA and discover the optimum length of asRNA. </li><br />
</ul><br />
<p><br />
Our project provides not only high efficient asRNAs but also brand new viewpoints to your project.<br />
As yet, almost all the projects presented to iGEM have focused to regulate the behavior of <i>E. coli</i>, only by transforming plasmids.<br />
But now, you can use our asRNA and regulate expression of the genes which are on genome.<br />
</p><br />
<p><br />
We used hybridizing of mRNA and specific asRNA to resemble love of RNA, and named our project “RNA in Love” <br />
Let's watch over RNA in love!<br />
</p><br />
<br />
<br />
<br />
<br><br />
<br><br />
<ol class="citation-list"><br />
<li id="cite-1">C Claudia<i>et al.</i> (2012) Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491: 7424 454-</li><br />
<li id="cite-2">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
</ol><br />
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{{Team:HokkaidoU_Japan/JS}}</div>TaKeZohttp://2014.igem.org/Team:HokkaidoU_Japan/Projects/Overview/BackgroundTeam:HokkaidoU Japan/Projects/Overview/Background2014-10-17T16:40:48Z<p>TaKeZo: </p>
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<h1>Background</h1><br />
<p><br />
Discovering of non-coding RNA (ncRNAs) and reverse transcription of virus composes the central dogma that was declared by Francis Click. <br />
Since ncRNAs do not encode any proteins, they had been thought that they don't have any functions.<br />
However, it was proved that they do have many functions.<br />
For example, some ncRNAs reduce the efficiency of translation, while others promote that. (C. Claudia <i>et al.</i>, 2012<sup><a href="#cite-1">[1]</a></sup>). <br />
</p> <br />
<p><br />
There are ncRNAs called anti-sense RNA (asRNAs), which down-regulate translation. An asRNA has complement sequence of its target mRNA, and reduces the translation efficiency by hybridizing. <br />
asRNAs can be easily synthesized, but there is no clear method to make stable, highly efficient asRNAs. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/9/93/HokkaidoU_Background01.png"><br />
<div><br />
Fig. 1 An asRNA hybridizes with target mRNA and interrupts entry of ribosome.<br />
</div><br />
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<br />
<div class="fig fig400"><br />
<img style="width:300px;" src="https://static.igem.org/mediawiki/2014/f/fd/HokkaidoU_Hstem_RNA.png"><br />
<div><br />
Fig. 2 Stem structure stabilizes asRNA<br />
</div><br />
</div><br />
<br />
<p><br />
In this situation, Nakashima and his colleagues discovered asRNA that is sandwiched by 38 base pairs of repeated sequence is stable and regulates translation at a high efficiency (N Nakashima <i>et al.</i>, 2006<sup><a href="#cite-2">[2]</a></sup>).<br />
</p><br />
<p><br />
Many iGEM teams prepare their project using asRNA, but how many teams use the asRNA that is based on a clear design concept?<br />
This year, we provide you a BioBrick standardized asRNA.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<p><br />
Our procedure to design asRNAs will assure you that<br />
</p><br />
<ul><br />
<li>The asRNAs are based on a clear design concept.</li><br />
<li>They are calculated to have ideal second structures.</li><br />
<li>They have high efficiency to regulate translation, based on our results. </li><br />
</ul><br />
<p><br />
By using those asRNAs, we did the projects below.<br />
</p><br />
<ul><br />
<li>Construct our asRNA that has modified stem sequence.</li><br />
<li> Construct B0034 -most popular RBS- specific asRNA, and regulate translation efficiency generally.</li><br />
<li> Construct various length asRNA and discover the optimum length of asRNA. </li><br />
</ul><br />
<p><br />
Our project provides not only high efficient asRNAs but also brand new viewpoints to your project.<br />
As yet, almost all the projects presented to iGEM have focused to regulate the behavior of <i>E. coli</i>, only by transforming plasmids.<br />
But now, you can use our asRNA and regulate expression of the genes which are on genome.<br />
</p><br />
<p><br />
We used hybridizing of mRNA and specific asRNA to resemble love of RNA, and named our project “RNA in Love” <br />
Let's watch over RNA in love!<br />
</p><br />
<br />
<br />
<br />
<br><br />
<br><br />
<ol class="citation-list"><br />
<li id="cite-1">C Claudia<i>et al.</i> (2012) Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat. Nature 491: 7424 454-</li><br />
<li id="cite-2">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
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<h1 id="Overview">Overview</h1><br />
<p><br />
The weak point of silencing by anti-sense RNA (asRNA) is the instability of RNA. RNA has a shorter half-life than DNA, so it is essential for asRNA to be stabilized to repress mRNA sufficiently. This problem was solved by inserting each-complementary sequeces called stem regions into upstream and downstream of as RNA (N Nakashima <i>et al.</i>, 2006<sup><a href="#cite-1">[1]</a></sup>). These regions make asRNA form stem-loop structure, which leads to increase the stability of asRNA dramatically.<br />
</p><br />
<br />
<p><br />
We followed in their footsteps. We designed our unique stem sequence named H-stem and producecd a BioBricked vector for asRNA expression so that other iGEMers and researchers can easily use stemed asRNA. Using this vector, you can gain an asRNA-expressing construct just by inserting PCRed target gene into the vector.<br />
</p><br />
<br />
<br />
<h1 id="asRNA_stabilization">asRNA stabilization by stem structure</h1><br />
<br />
<p><br />
The stem regions flank asRNA region as shown in Fig. 1 on DNA. <br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/f/f7/HokkaidoU_Hstem_DNA.png"><br />
<div>Fig. 1 stem and anti-sense region on DNA</div><br />
</div><br />
<br />
<p><br />
Transcripted to RNA, stem sequences bind each other and make the whole RNA form stem-loop structure like Fig. 2. <br />
</p><br />
<br />
<div class="fig fig400"><br />
<img src="https://static.igem.org/mediawiki/2014/f/fd/HokkaidoU_Hstem_RNA.png"><br />
<br />
<div>Fig. 2 stem and anti-sense region on RNA</div><br />
</div><br />
<p><br />
This stem-loop structure contributes to RNA stabilization on the following two points.<br />
</p><br />
<br />
<p><br />
One is the protection from the exonuclease activity of RNase. Since RNase in the cell decomposes single-strand RNA, stemed asRNA is hardly affected by such a degradation.<br />
</p><br />
<br />
<p><br />
The other is an increase of thermodynamical stability by a decrease of Gibbs energy. Stem region produces such a great amount of negative $\Delta G$ that positive $\Delta G$ produced by the curvature of loop region is overwhelmed.<br />
</p><br />
<div class="clearfix"></div><br />
<br />
<br />
<br />
<br />
<br />
<p><br />
In bacterial cells, the decomposability of asRNA to that of target mRNA affects the amount of translated mRNA as shown below. Here, x-axis indicates the expression level of asRNA to that of mRNA.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/5/55/HokkaidoU_Hstem_graph.png"><br />
</div><br />
<br />
<p><br />
From that graph, you can see that the stability of asRNA greatly contributes to the repression of mRNA. For the detail, please refer to the following.<br />
</p><br />
<br />
<br />
<h2 id="Modelling"><div id="button">Modelling&nbsp;<i class="fa fa-angle-double-down"></i></div></h2><br />
<br />
<br />
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<div id="text"><!--モデリングで隠せるのはここから--><br />
<br />
<p><br />
The reaction of asRNA (hereinafter referred to as $X$), mRNA ($Y$) and RNA duplex ($Z$) consisting of the asRNA and the mRNA in a bacterial cell is<br />
</p><br />
<br />
\[ X+Y \overset{k_{\rm bind}}{\underset{k_{\rm unbind}}{\rightleftharpoons}} Z \]<br />
<br />
<p><br />
Besides, mRNA and asRNA are produced and decomposed at all times. So, the ordinary defferential equation that represets the whole reaction in the cell is shown below.<br />
</p><br />
<br />
\begin{cases}<br />
\dot{x}=a-bx-k_{\rm bind}xy+k_{\rm unbind}z & \\<br />
\dot{y}=1-y-k_{\rm bind}xy+k_{\rm unbind}z & \\<br />
\dot{z}=k_{\rm bind}xy-k_{\rm unbind}z-cz &<br />
\end{cases}<br />
<br />
<p><br />
Here, we took $1$ for two constants of the right side first and second terms in the second formula using the flexibilities of $[time]$ and $[concentration]$. Thus, constant $a$ stands for the expression level of asRNA to that of target mRNA and constant $b$ the decomposability of asRNA to that of mRNA. <br />
</p><br />
<br />
<p><br />
The unique stable fixed point (in quadrant I) of the ODE is easily found by taking $0$ for each equation. Especially, $y$ value of the fixed point, $y^*$, is<br />
</p><br />
<br />
\[ y=\frac{1}{2} \biggl\{ \sqrt{ \bigl( a-1+\frac{b}{\gamma} \bigl)^2 +4 \frac{b}{\gamma}} - \Bigl( a-1+\frac{b}{\gamma} \Bigl) \biggl\} \]<br />
<br />
<p><br />
Provided that \[ \gamma = \frac{k_{\rm bind}c}{k_{\rm unbind}+c} \]<br />
</p><br />
<br />
<p><br />
We find the graph above by regarding this formula as a function of $a$.<br />
</p><br />
<br />
<p><br />
Though here we let $\gamma$ value be $1$ arbitarily, this value does not affect qualitatively as the formula shows.<br />
</p><br />
</div><!--ここまで!!!!!!--><br />
<br />
<br />
<h1 id="Construct">Construct</h1><br />
<p><br />
The construct which expresses this H-stem system, named H-stem vector, has such a sequence as shown below.<br />
</p><br />
<br />
<div class="fig fig800"><br />
<img src="https://static.igem.org/mediawiki/2014/b/b9/HokkaidoU_Hstem_construct.png"><br />
<div>Fig. 3 The structure of H-stem system</div><br />
</div><br />
<br />
<br />
<p><br />
Stemed asRNA is expressed by a reliable inducible promoter, P<sub>lac</sub>, and it is activated by IPTG induction.<br />
</p><br />
<br />
<p><br />
Since anti-sense region can be digested out by NcoI and XhoI, you can insert any anti-sense sequence as recounted below. Aditionally, a dummy anti-sense sequence is in advance inserted into this vector.<br />
</p><br />
<br />
<p><br />
Notice that this stem sequence has a resistance to PCR, especially sequencing PCR. If you want to sequence it, you have to devide it into upstream part and downstream part before PCR. (We suppose that single-strand DNA of this sequence also forms stem-loop structure and it prevents from the extension of complementary strand, but the detailed machanism is unknown.) <br />
</p><br />
<br />
<h1 id="How_To_Use">How to Use</h1><br />
<br />
<p><br />
Now, we explain how to use H-stem vector.<br />
</p><br />
<br />
<p><br />
What you need is two restriction enzymes, NcoI and XhoI, and two PCR primers, added to the H-stem vector.<br />
</p><br />
<br />
<br><br />
<br />
<ol style="display:block;" type="1"><br />
<li>Determine the asRNA binding sequence, which is about 60 bases, from the tail of RBS to the head of CDS. (Please confirm that the sequence does not include NcoI or XhoI site.)</li><br />
<li>Design the primers which amplify the sequence. Here, add XhoI recognition site (5’-GGG<b>CTCGAG</b>…) to 5’ end of the forward primer and NcoI recognition site (5’-GGG<b>CCATGG</b>…) to that of forward primer.</li><br />
<li>PCR the sequence by the two primers.</li><br />
<li>Digest the PCR product and H-stem vector by NcoI and XhoI, mix them, and ligate together.</li><br />
</ol><br />
<br />
<p><br />
We hope that this method will be a help to many iGEMers.<br />
</p><br />
<br />
<br><br />
<br><br />
<h3>Reference</h3><br />
<ol class="citation-list"><br />
<li id="cite-1">N Nakashima <i>et al.</i> (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in <i>Escherichia coli</i>. Nucleic Acids Res 34: 20 e138</li><br />
</ol><br />
<br />
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-moz-transform: rotateX(0deg);<br />
transform: rotateX(0deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-vertical .bb-flip-next-end {<br />
-webkit-transform: rotateY(-15deg);<br />
-moz-transform: rotateY(-15deg);<br />
transform: rotateY(-15deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-vertical .bb-flip-prev-end {<br />
-webkit-transform: rotateY(-165deg);<br />
-moz-transform: rotateY(-165deg);<br />
transform: rotateY(-165deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-horizontal .bb-flip-next-end {<br />
-webkit-transform: rotateX(15deg);<br />
-moz-transform: rotateX(15deg);<br />
transform: rotateX(15deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-horizontal .bb-flip-prev-end {<br />
-webkit-transform: rotateX(165deg);<br />
-moz-transform: rotateX(165deg);<br />
transform: rotateX(165deg);<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
.bb-item {<br />
width: 100%;<br />
position: absolute;<br />
top: 0;<br />
left: 0;<br />
display:none;<br />
font-family: 'Special Elite', cursive;<br />
}<br />
<br />
/* No JS */<br />
.no-js .bb-bookblock, <br />
.no-js ul.bb-custom-grid li {<br />
width: auto;<br />
height: auto;<br />
}<br />
<br />
.no-js .bb-item {<br />
display: block;<br />
position: relative;<br />
}<br />
<br />
<br />
.bb-custom-wrapper {<br />
width: 960px;<br />
position: relative;<br />
margin: 0 auto;<br />
text-align: center;<br />
}<br />
<br />
.bb-custom-wrapper .bb-bookblock {<br />
/*box-shadow: 0 12px 20px -10px rgba(81,64,49,0.6);*/<br />
overflow: hidden;<br />
}<br />
<br />
.bb-custom-wrapper h3 {<br />
font-size: 1.4em;<br />
font-weight: 300;<br />
margin: 0.4em 0 1em;<br />
}<br />
<br />
.bb-custom-wrapper nav {<br />
width: 100%;<br />
height: 30px;<br />
margin: 1em auto 0;<br />
position: relative;<br />
z-index: 0;<br />
text-align: center;<br />
}<br />
<br />
.bb-custom-wrapper nav a {<br />
display: inline-block;<br />
width: 30px;<br />
height: 30px;<br />
text-align: center;<br />
border-radius: 2px;<br />
margin: 2px;<br />
}<br />
<br />
.bb-custom-wrapper nav a:hover {<br />
opacity: 0.6;<br />
}<br />
<br />
<br />
<br />
.left-page-top {<br />
float: left;<br />
width:49%;<br />
height: auto;<br />
position: absolute;<br />
top:50px;<br />
}<br />
<br />
.left-page {<br />
float: left;<br />
width:49%;<br />
height: auto;<br />
position: absolute;<br />
top:0;<br />
}<br />
<br />
.right-page-top {<br />
float: left;<br />
width: 50%;<br />
height: 100%;<br />
position:absolute;<br />
left:50%;<br />
text-align:left;<br />
}<br />
<br />
.right-page {<br />
float: left;<br />
width: 49%;<br />
height: auto;<br />
position:absolute;<br />
top:0;<br />
text-align:left;<br />
}<br />
<br />
.page-title {<br />
margin: 30px auto;<br />
font-size: 40px;<br />
font-weight: bold;<br />
font-family: 'Special Elite', cursive;<br />
position:relative;<br />
top:-600px;<br />
}<br />
<br />
.book-index {<br />
text-align: left;<br />
margin: 20px auto;<br />
list-style: none;<br />
position: absolute;<br />
}<br />
<br />
.book-index > ul > li {<br />
font-size: 25px;<br />
margin: 20px auto;<br />
list-style: none;<br />
}<br />
<br />
.book-index ul {<br />
list-style: none;<br />
line-height:30px;<br />
}<br />
<br />
.book-index > ul > li > ul > li {<br />
font-size: 15px;<br />
list-style: none;<br />
}<br />
<br />
.bb-item-contents {<br />
font-size: 30px;<br />
line-height: 60px;<br />
margin: 25px 100px ;<br />
position: absolute;<br />
}<br />
<br />
.bb-item-picture {<br />
position: relative;<br />
right: -300px;<br />
z-index:101;<br />
}<br />
<br />
.top-picture {<br />
width: 100%;<br />
height: 100%;<br />
}<br />
<br />
.book-background {<br />
width:100%;<br />
height:100%;<br />
position: relative;<br />
}<br />
<br />
.page-vertical-top {<br />
height:300px;<br />
width:100%;<br />
position:absolute;<br />
top:0px;<br />
}<br />
<br />
.page-vertical-bottom {<br />
height:300px;<br />
width:100%;<br />
position:absolute;<br />
top:300px;<br />
}<br />
.page-picture {<br />
width:270px;<br />
position:relative;<br />
}<br />
<br />
.left-up {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 20px;<br />
top:0px;<br />
}<br />
<br />
.right-up {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 10px;<br />
top:0px;<br />
}<br />
<br />
.left-bottom {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 20px;<br />
top:-21px;<br />
}<br />
<br />
.right-bottom {<br />
float:left;<br />
width:200px;<br />
position: relative;<br />
left:100%;<br />
margin:0 10px;<br />
top:-21px;<br />
}<br />
<br />
.character-pic {<br />
width:145px;<br />
position:relative;<br />
left:20px;<br />
top:5px;<br />
}<br />
<br />
.picture-fin {<br />
width:350px;<br />
position:relative;<br />
left:500px;<br />
<br />
}<br />
<br />
.picture-last {<br />
width:70%;<br />
position:relative;<br />
top:150px;<br />
}<br />
<br />
.index-left {<br />
float:left;<br />
width:50%;<br />
position:absolute;<br />
top:60px;<br />
left:100px;<br />
}<br />
<br />
.index-right {<br />
float:left;<br />
width:50%;<br />
position:absolute;<br />
left:530px;<br />
top:60px;<br />
}<br />
<br />
.text-box {<br />
width:75%;<br />
margin:130px auto;<br />
font-size:20px;<br />
}<br />
<br />
.text-box > p {<br />
line-height:45px;<br />
}<br />
<br />
.bb-custom-wrapper nav {<br />
position: absolute;<br />
top: 0; left: -100px;<br />
margin: 0;<br />
padding: 0;<br />
width: 1160px;<br />
height: 600px;<br />
z-index: 1000;<br />
}<br />
<br />
#bb-nav-next {<br />
position: absolute;<br />
top: 0; right: 0;<br />
width: 580px;<br />
height: 600px;<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
#bb-nav-prev {<br />
position: absolute;<br />
top: 0; left: 0;<br />
width: 580px;<br />
height: 900px;<br />
margin: 0;<br />
padding: 0;<br />
}<br />
<br />
#bb-nav-next i {<br />
position: absolute;<br />
top: 480px; right: 50px;<br />
margin: -18px -6px 0 0;<br />
padding: 0;<br />
}<br />
<br />
#bb-nav-prev i {<br />
position: absolute;<br />
top: 480px; left: 50px;<br />
margin: -18px 0 0 -6px;<br />
padding: 0;<br />
}<br />
<br />
.bb-page,<br />
.bb-item,<br />
.left-page-top,<br />
.right-page-top,<br />
.bb-flip-prev,<br />
.bb-flip-next,<br />
.bb-flip-prev-end,<br />
.bb-flip-next-end,<br />
.bb-flip-initial,<br />
.bb-front,<br />
.bb-back,<br />
.bb-flipoverlay<br />
{<br />
height: 600px !important;<br />
}<br />
<br />
.bb-item,<br />
.bb-page {<br />
margin: 180px 0;<br />
}<br />
<br />
/* No JS */<br />
.no-js .bb-custom-wrapper {<br />
height: auto;<br />
}<br />
<br />
.no-js .bb-custom-content {<br />
height: 470px;<br />
}<br />
<br />
<br />
</style><br />
</html></div>TaKeZo