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http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:45:18Z
<p>PengYong: </p>
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<h1 style="border:none;" id="goal">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
<br/><br />
<br/><br />
<h1 style="border:none;" id="design">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
<br/><br />
<br/><br />
<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
<br/><br />
<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
<br/><br />
<br/><br />
NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
<br/><br />
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<br/><br />
<h2 id="platform">Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
<br/><br />
<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
<br/><br />
<br/><br />
<h2 id="theoretical">Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
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<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
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<br/><br />
<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
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<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
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<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
<br/><br />
<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
<br/><br />
<br/><br />
<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
<br/><br />
<br/><br />
<h2 id="rna">RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
<br/><br />
<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
<br/><br />
<p>The modular architecture of artificial small RNA are as follows:<br />
<br/><br />
<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
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<br/><br />
<h2 id="comparator">Artificial Region Design: Comparator</h2><br />
<br/><br />
<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
<br/><br />
<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
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<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
<br/><br />
<br/><br />
<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
<br/><br />
<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
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<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
<br/><br />
<br/><br />
<h2 id="sensor">Artificial Region Design: Ratio sensor</h2><br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
<br/><br />
<br/><br />
<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
<br/><br />
<br/><br />
<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
<br/><br />
<br/><br />
<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
<br/><br />
<br/><br />
<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
<br />
<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
<br />
<br/><br />
<br/><br />
<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
<br/><br />
<br/><br />
<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
<br/><br />
<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
<br/><br />
<br/><br />
<h2 id="region">Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
<br/><br />
<br/><br />
<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
<br/><br />
<br/><br />
<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
<br/><br />
<br/><br />
<p><strong>Project flow chart</strong><br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/f/f3/Ouc-project-designmaking-design20.JPG" /></a><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:32:03Z
<p>PengYong: </p>
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<h1 style="border:none;" id="goal">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
<br/><br />
<br/><br />
<h1 style="border:none;" id="design">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
<br/><br />
<br/><br />
<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
<br/><br />
<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
<br/><br />
<br/><br />
NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
<br/><br />
<br/><br />
<h2 id="platform">Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
<br/><br />
<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
<br/><br />
<br/><br />
<h2 id="theoretical">Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
<br/><br />
<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
<br/><br />
<br/><br />
<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
<br/><br />
<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
<br/><br />
<br/><br />
<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
<br/><br />
<br/><br />
<h2 id="rna">RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
<br/><br />
<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
<br/><br />
<p>The modular architecture of artificial small RNA are as follows:<br />
<br/><br />
<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
<br/><br />
<br/><br />
<h2 id="comparator">Artificial Region Design: Comparator</h2><br />
<br/><br />
<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
<br/><br />
<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
<br/><br />
<br/><br />
<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
<br/><br />
<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
<br/><br />
<br/><br />
<h2 id="sensor">Artificial Region Design: Ratio sensor</h2><br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
<br/><br />
<br/><br />
<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
<br/><br />
<br/><br />
<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
<br/><br />
<br/><br />
<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
<br/><br />
<br/><br />
<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
<br />
<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
<br />
<br/><br />
<br/><br />
<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
<br/><br />
<br/><br />
<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
<br/><br />
<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
<br/><br />
<br/><br />
<h2 id="region">Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
<br/><br />
<br/><br />
<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
<br/><br />
<br/><br />
<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
<br/><br />
<br/><br />
<p><strong>Project flow chart</strong><br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/f/f3/Ouc-project-designmaking-design20.JPG" /></a><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:25:17Z
<p>PengYong: </p>
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<div class="list-box"><br />
<div class="list-text"><br />
<h1 style="border:none;" id="goal">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
<br/><br />
<br/><br />
<h1 style="border:none;" id="design">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
<br/><br />
<br/><br />
<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
<br/><br />
<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
<br/><br />
<br/><br />
NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
<br/><br />
<br/><br />
<h2 id="platform">Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
<br/><br />
<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
<br/><br />
<br/><br />
<h2>Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
<br/><br />
<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
<br/><br />
<br/><br />
<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
<br/><br />
<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
<br/><br />
<br/><br />
<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
<br/><br />
<br/><br />
<h2>RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
<br/><br />
<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
<br/><br />
<p>The modular architecture of artificial small RNA are as follows:<br />
<br/><br />
<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
<br/><br />
<br/><br />
<h2>Artificial Region Design: Comparator</h2><br />
<br/><br />
<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
<br/><br />
<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
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<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
<br/><br />
<br/><br />
<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
<br/><br />
<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
<br/><br />
<br/><br />
<h2>Artificial Region Design: Ratio sensor</h2><br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
<br/><br />
<br/><br />
<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
<br/><br />
<br/><br />
<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
<br/><br />
<br/><br />
<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
<br/><br />
<br/><br />
<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
<br />
<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
<br />
<br/><br />
<br/><br />
<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
<br/><br />
<br/><br />
<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
<br/><br />
<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
<br/><br />
<br/><br />
<h2>Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
<br/><br />
<br/><br />
<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
<br/><br />
<br/><br />
<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
<br/><br />
<br/><br />
<p><strong>Project flow chart</strong><br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/f/f3/Ouc-project-designmaking-design20.JPG" /></a><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:19:40Z
<p>PengYong: </p>
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<div class="mini-riboon"><img src="https://static.igem.org/mediawiki/2012/0/03/Ouc-mini-ribbon-decision-making.png"></div><br />
<div class="article-list"><br />
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<div class="list-text"><br />
<h1 style="border:none;">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
<br/><br />
<br/><br />
<h1 style="border:none;">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
<br/><br />
<br/><br />
<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
<br/><br />
<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
<br/><br />
<br/><br />
NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
<br/><br />
<br/><br />
<h2>Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
<br/><br />
<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
<br/><br />
<br/><br />
<h2>Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
<br/><br />
<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
<br/><br />
<br/><br />
<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
<br/><br />
<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
<br/><br />
<br/><br />
<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
<br/><br />
<br/><br />
<h2>RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
<br/><br />
<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
<br/><br />
<p>The modular architecture of artificial small RNA are as follows:<br />
<br/><br />
<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
<br/><br />
<br/><br />
<h2>Artificial Region Design: Comparator</h2><br />
<br/><br />
<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
<br/><br />
<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
<br/><br />
<br/><br />
<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
<br/><br />
<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
<br/><br />
<br/><br />
<h2>Artificial Region Design: Ratio sensor</h2><br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
<br/><br />
<br/><br />
<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
<br/><br />
<br/><br />
<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
<br/><br />
<br/><br />
<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
<br/><br />
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<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
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<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
<br />
<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
<br />
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<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
<br/><br />
<br/><br />
<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
<br/><br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
<br/><br />
<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
<br/><br />
<br/><br />
<h2>Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
<br/><br />
<br/><br />
<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
<br/><br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
<br/><br />
<br/><br />
<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
<br/><br />
<br/><br />
<p><strong>Project flow chart</strong><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:18:53Z
<p>PengYong: </p>
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<h1 style="border:none;">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
</p><br />
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<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
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<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
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<h1 style="border:none;">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
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<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
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<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
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<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
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NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
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<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
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<h2>Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
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<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
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<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
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<h2>Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
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<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
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<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
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<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
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<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
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<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
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<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
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<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
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<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
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<h2>RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
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<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
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<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
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<p>The modular architecture of artificial small RNA are as follows:<br />
<br/><br />
<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
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<h2>Artificial Region Design: Comparator</h2><br />
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<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
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<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
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<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
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<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
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<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
<br/><br />
<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
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<h2>Artificial Region Design: Ratio sensor</h2><br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
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<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
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<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
<br/><br />
<br/><br />
<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
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<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
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<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
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<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
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<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
<br />
<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
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<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
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<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
<br/><br />
<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
<br/><br />
<br/><br />
<h2>Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
<br/><br />
<br/><br />
<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
<br/><br />
<br/><br />
<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
<br/><br />
<br/><br />
<p><strong>Project flow chart</strong><br />
<br/><br />
<br/><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:12:49Z
<p>PengYong: </p>
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<h1 style="border:none;">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
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<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
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<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
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<h1 style="border:none;">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
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<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
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<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
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<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
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NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
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<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
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<h2>Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
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<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
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<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
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<h2>Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
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<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
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<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
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<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
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<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
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<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
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<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
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<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
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<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
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<h2>RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
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<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
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<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
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<p>The modular architecture of artificial small RNA are as follows:<br />
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<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
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<h2>Artificial Region Design: Comparator</h2><br />
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<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
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<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
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<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
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<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
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<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
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<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
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<h2>Artificial Region Design: Ratio sensor</h2><br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
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<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
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<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
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<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
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<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
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<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
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<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
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<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
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<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
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<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
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<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
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<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
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<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
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<h2>Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
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<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
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<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
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<p><strong>Project flow chart</strong><br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/f/f3/Ouc-project-designmaking-design20.JPG" /></a><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:12:02Z
<p>PengYong: </p>
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<li id="menu_design"><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign" title="Goal&Design" class="design">Goal&Design</a></li><br />
<li id="menu_others"><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result" title="Result" class="others">Result</a></li><br />
<li id="menu_books"><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/DiscussionandFuture" title="Discussion" class="books">Discussion</a></li><br />
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<div class="mini-riboon"><img src="https://static.igem.org/mediawiki/2012/0/03/Ouc-mini-ribbon-decision-making.png"></div><br />
<div class="article-list"><br />
<div class="start"></div><br />
<div class="list-box"><br />
<div class="list-text"><br />
<h1 style="border:none;">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
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<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
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<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
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<h1 style="border:none;">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
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<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
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<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
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<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
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NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
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<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
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<h2>Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
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<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
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<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
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<h2>Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
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<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
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<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
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<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
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<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
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<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
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<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
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<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
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<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
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<h2>RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
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<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
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<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
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<p>The modular architecture of artificial small RNA are as follows:<br />
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<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
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<h2>Artificial Region Design: Comparator</h2><br />
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<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
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<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
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<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
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<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
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<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
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<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
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<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
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<h2>Artificial Region Design: Ratio sensor</h2><br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
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<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
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<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
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<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
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<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
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<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
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<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
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<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
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<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
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<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
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<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
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<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
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<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
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<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
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<h2>Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
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<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
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<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
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<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
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<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
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<p><strong>Project flow chart</strong><br />
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<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/f/f3/Ouc-project-designmaking-design20.JPG" /></a><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign
Team:OUC-China/Project/DesignMaking/GoalandDesign
2012-10-27T03:11:13Z
<p>PengYong: </p>
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<h1 style="border:none;">Goal</h1><br />
<p><br />
<a><img style="margin-top:5px;" src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-designmaking-goal0.jpg" /></a><br />
<p style="margin-left:250px; margin-top: -70px;">We are going to construct a trial device to process two different inputs(eg.aTc,IPTG) into detectable fluorescence change.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/6/6d/Ouc-project-designmaking-goal1.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>After constructing the trial device,we will assembly the fine-tuned Nitrate/Phosphate sensors and Gas Vesicle generator to a well characterized decision-making device,to make a full version Red Tide Alarmer.<br />
<br/><br />
<br/><br />
<h1 style="border:none;">Design</h1><br />
<p><strong>Insights into the “black box”</strong><br />
<span></span>Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
<br/><br />
<br/><br />
<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.1 Insights into the black box:<br />
“[ ]” stands for concentration;<br />
“k” stands for RNA reaction constant<br />
</p><br />
<br/><br />
<p><strong>Decision-making device(trial) final construct</strong><br />
<span></span>Tunable promoter Ptet/Plac controls the transcription rate(αs,nM/min) of two small RNAs;we expect the aTc/IPTG gradient will generate different small RNA levels in cytosol,which could be monitored by our GFP generator that has been fused to sRNA target site.<br />
For Ratio Sensor,we selected a small RNA scaffold which would fuse artificial region to 5’ end of small RNA to construct a strong sRNA::sRNA interaction,the sRNA buffer with each other in cytosol in this case;<br />
For comparator,km2=0,which means sRNA2 is a pool of buffer for sRNA1,at the same time establish a tunable threshold for comparing quantitatively.<br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design1.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.2 Final construct of our trial decision-making device.<br />
<br/><br />
<br/><br />
NOTE:several independent units in this figure maybe assembly on different copy number plasmids for double-plasmids transformation<br />
</p><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/6/6b/Ouc-project_overview.png" /></a><br />
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<br/><br />
<h2>Platform Construction <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We construct tet/lac_GFP generator below to characterize R0011(Plac) and R0040(Ptet) promoting behaviour,so that we could alter the sRNA levels in cytosol relatively.<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a><br />
<br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/2/26/Ouc-project-designmaking-design2.JPG" /></a><br />
<p style="text-align:center; font-size:90%;">Fig.3 sRNA assembly platform.<br />
<br/><br />
<br/>E0240 is a classic GFP translational unit with weak RBS to characterize the promoter behaviour<br />
</p><br />
<br/><br />
<br/><br />
<h2>Theoretical analysis through modeling</h2><br />
<p><strong>What’s in the 3-RNA system?</strong><span></span>We formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
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<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
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<a><img src="https://static.igem.org/mediawiki/2012/9/9d/Ouc-project-designmaking-design5.jpg" /></a><br />
<br/><br />
<br/><br />
<p><strong>Is it feasible?</strong><br />
<span></span>In this three-ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell,and it’s apparent enough considering stochasticity.<br />
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<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Subsquent analysis</strong><span></span>We conducted a theoretical analysis consists of four steps(<a https://2012.igem.org/Team:OUC-China/Modeling/Overview<br />
">Modeling Section</a>)<br />
<br/><br />
<br/><a><img src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-designmaking-design7.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>The modeling analysis shed some light on small RNA scaffold selection:in this three-ternary system,one thing is clear, km is the most important parameter in this system in given parameter set,if km is not low enough(>1),there won’t be any chance to get the ideal behaviours by fine-tuning other accessible parameters like the range of transcription rate(α) and sRNA::sRNA binding rate(ks).<br />
<br/><br />
<br/><br />
<h2>sRNA scaffold selection:</h2><br />
<a><img src="https://static.igem.org/mediawiki/2012/7/77/Ouc-project-designmaking-design8.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.4 Fold regulation was calculated from the relative fluorescence by dividing the fluorescence obtained in the presence of the sRNA plasmid by that obtained with a control plasmid that does not express a regulatory RNA.<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span>We investigated many srNAs,finally selected <i>spot42</i> sRNA and galk::mRNA as our engineering chassis for several reasons:<br />
<br/>-1 Theoretical analysis has shown that the mRNA repression rate must be weak enough,we screened small RNAs from characterization data,found that <i>spot42</i> had the weakest repression capability;<br />
<br/>-2 <i>spot42</i> is a multitarget sRNA,which is favourable in screening comparator buffer RNA with varied affinity;<br />
<br/>-3 <i>spot42</i> has a weak RNA chaperone Hfq binding site,where as galK weaker,which is favourable in sRNA::sRNA interaction with Hfq;<br />
<br/>-4 It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.<i>spot42</i>-galK complex degrades in a slow and presently unclear way;<br />
<br/>-5 Well studied,data is sufficient enough.<br />
<br/><br />
<br/><br />
<h2>RNA Scaffold and Platform assembly and quantitative tests <a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">(experimental flow chart)</a></h2><br />
<p><span></span>We ligate RNA scaffold and previous platform together ,then tested them quantitatively by plate reader, characterize the relationship between inducer concentration gradient and <i>spot42</i>-galK::GFP repression efficiency.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/7/71/Ouc-project-designmaking-design9.JPG" /></a><br />
<br/><br />
<p><a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">See the data!</a><br />
<br/><br />
<p>The modular architecture of artificial small RNA are as follows:<br />
<br/><br />
<a><img style="margin-left:130px;" src="https://static.igem.org/mediawiki/2012/9/91/Ouc-project-designmaking-design10.JPG" /></a><br />
<a href="https://static.igem.org/mediawiki/2012/3/3d/Ouc-zp1.jpg">Details </a><br />
<br/><br />
<br/><br />
<h2>Artificial Region Design: Comparator</h2><br />
<br/><br />
<a><img style="margin-left:8px;" src="https://static.igem.org/mediawiki/2012/5/5f/Ouc-project-designmaking-design11.JPG" /></a><br />
<br/><br />
<p><strong>1.Modeling analysis</strong><br />
<span></span>As we have mentioned, modeling analysis tells us that interactions between comparator buffer RNA(targets of <i>spot42</i>) and <i>spot42</i> should be far stronger than galK::gfp transcript and <i>spot42</i>. To this end, we need to make in-depth investigations in <i>spot42</i>-mediated gene silencing.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>2.<i>spot42</i> targets investigation</strong><br />
<span></span>Fortunately Chase L. Beisel has made detailed works about the network that <i>spot42</i> is involved in catabolite repression. Following microarray analysis and beta-galactosidase assays for LacZ-fusion reported by Chase L. Beisel, we finally selected three <i>spot42</i> targets, <i>nanC</i>, <i>ytfJ</i> and <i>srlA</i> as our candidates for comparator buffer RNA. Here are our candidates’ results of microarray analysis.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9a/Ouc-project-designmaking-design12.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.5 The ratio of mRNA levels for pBRplac and p<i>spot42</i> samples is shown at the right end. We could see that the repression rates of all our candidates are more than 2.6-fold, higher than galK. Although <i>nanC</i> does not work well in microarray analysis, the author indicated that <i>nanC</i>::lacZ fusion gives surprisingly more than 47-fold repression rate which seems to match our requirement well.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.</p><br />
<br/><br />
<br/><br />
<p><strong>3.Analysis secondary structure & binding sites of target mRNA</strong><br />
<span></span>Next we considered if there are motifs that facilitate the interaction by means of some RNA prediction software such as RNA structure, intaRNA and NUPACK.<br />
<br/><br />
<span></span>After confirming the interactive mechanisms, we decided to construct a trial platform to verify the feasibility of our design and acquire the first-hand data for further fine-tuning. The brief introduction of our comparator trial device is below.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">Learn more</a>.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/e/e4/Ouc-project-designmaking-design13.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.6 our design for double plasmids inspection.On plasmid J61002, P0440 can produce repressor TetR to repress R0040 while P0412 can produce repressor LacI to repress R0011.The Repression caused by TetR and LacI can be offset by aTc and IPTG repectively, resulting in transcription of buffer RNA and <i>spot42</i>. The other plasmid on the left side contains a reporter gene GFP, fusing with galK. Provided that concentration of <i>spot42</i> in cytosol surpasses that of buffer RNA, it will act as galK::GFP inhibitor to repress the expression of GFP</p><br />
<br/><br />
<br/><br />
<h2>Artificial Region Design: Ratio sensor</h2><br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/9/9c/Ouc-project-designmaking-design14.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Modeling analysis</strong><span></span>Similar to our comparator design, we first adjusted our design ideas according to the model we construct. It indicates that ks should be far higher than km which enables sRNA-sRNA interactions prior to sRNA-mRNA interactions.<a href="https://2012.igem.org/Team:OUC-China/Project/Supplementary">See more</a>.<br />
<br/><br />
<br/><br />
<p><strong>sRNA investigation</strong><br />
<span></span>To this end, we have compared a lot of sRNA-mRNA interactions, both trans-acting and cis-acting. Unfortunately, few literatures tried to make comparison between different RNA expression rates for artificial design, let alone the construction of ternary RNAs system. So we have worked extremely hard to shape and improve our design ideas. <br />
<br/><br />
<br/><br />
<p><strong>In silico analysis of 2 different design guidelines</strong><br />
<span></span>First, we searched for sRNA-mRNA interactive mechanisms that show potential for relatively strong interaction. We have brainstormed several ideas in which we picked up only one that showed great potential for success. We singled out translational activated reaction of <i>dsrA</i>-<i>rpos</i> as our ‘raw materials’ for artificial region. It works like this: <i>dsrA</i> activates <i>rpos</i> translation by base-pairing with the 5’ <i>rpos</i> leader, which relieves an intra-molecular stem-loop structure that sequesters the <i>rpos</i> ribosome binding site (RBS). The location of complementary pairing site of <i>dsrA</i>& <i>rpos</i> is shown below. <br />
<br/><br />
<br/><br />
<a><img style="margin-left:30px;" src="https://static.igem.org/mediawiki/2012/f/f4/Ouc-project-designmaking-design15.JPG" /></a><br />
<br/><br />
<br/><br />
<p>Secondary structure of <i>dsrA</i>&<i>rpos</i> as well as completed artificial sRNA(<i>dsrA</i>::spo42 & <i>rpos</i>::<i>spot42</i>) is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:55px" src="https://static.igem.org/mediawiki/2012/1/16/Ouc-project-designmaking-design16.JPG" /></a><br />
<br/><br />
<br/><br />
<p><a name="orthogonal"></a><div><strong>Orthogonal ratio sensor</strong></div><br />
<p><span></span>While our first design to construct a strengthened inert secondary structure of sRNA-sRNA is on progress, another design gradually matured, given that we have successfully constructed comparator which is based on multi-targets <i>spot42</i> network. The concept of this design focuses more on biological reaction mechanisms that blocking of RBS to prevent ribosomes loading on galK::GFP transcripts is necessary for GFP expression silencing. In view of this, it seems not reliable to simply look for non-cognated sRNA pairs to assembly to <i>spot42</i> respectively.<br />
<br />
<br/><span></span>Designs of comparator shed light on us that targets of <i>spot42</i> inserted into the AR(artificial region) might solve this problem. To this end, we have chosen <i>nanC</i> as an attempt for it. However, it is frustrating that when <i>nanC</i> with various lengths were assembled with <i>spot42</i> in silico, its secondary structure never failed to changed, leading to loss of functional domains. An orthogonal design finally solved this disturbing problem. Pair of mutant variant in <i>spot42</i> and complementary mutant variant in <i>nanC</i> have been reported previously and they worked perfectly as well. So we decided to introduce 5 bases mutant in <i>nanC</i>(<i>nanC</i>*) as well as complementary mutant in <i>spot42</i>(*<i>spot42</i>). Obviously <i>nanC</i>* is deprived of pairing with <i>spot42</i>, but given ability to pair with *<i>spot42</i>. It significantly protects its innate structures from self-repression when <i>nanC</i>*&<i>spot42</i>(or <i>nanC</i>&*<i>spot42</i> ) was assembled together. <br />
<br />
<br/><span></span>Moreover, fortunate as it is that complementary domain between <i>nanC</i> and <i>spot42</i> coincidently avoids that between galK and <i>spot42</i>, which prevent *<i>spot42</i> from deprivation of inhibition of galK::GFP expression. The complementary pairing of two pairs are shown below.<br />
<br />
<br/><br />
<br/><br />
<a><img style="margin-left:45px;" src="https://static.igem.org/mediawiki/2012/3/37/Ouc-project-designmaking-design17.JPG" /></a><br />
<br/><br />
<br/><br />
<p>The secondary structures of <i>spot42</i> , <i>nanC</i>, *<i>spot42</i>::<i>nanC</i> and <i>nanC</i>*::<i>spot42</i> is shown below.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/7/7b/Ouc-project-designmaking-design18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Here we can see that the structure is highly compatible when <i>nanC</i>*::<i>spot42</i> (or <i>nanC</i>::*<i>spot42</i>)assembled, involving previously described <i>dsrA</i>::<i>spot42</i>(or <i>rpos</i>::<i>spot42</i>). It further inspired us to carry out experiments to verify our designs.<br />
<br/><br />
<br/><p><strong>Future work</strong><span></span>Our experiment of those two designs are still in progress. Actually all the artificial sRNA assemblies have already succeeded, though have not been transformed into E.coli with combined plamids carrying our reporter genes. Provided that the-last-run transformation is finished, we could see whether our designs will work well or not. It is exciting that we would check them before the Asian Jamboree and showcase our results of our brand-new ‘ratio sensor’<br />
<br/><br />
<br/><br />
<h2>Artificial region assembly & Testing quantitatively</h2><br />
<p><strong>Assembly method</strong><br />
<span></span>We amplify artificial region by PCR from DH5α,using primers with ClaI/SalI site;<br />
ClaI site and SalI site are well-selected restriction sites,they are used to assembly the artificial region to the 5’ end of <i>spot42</i>,are compatible for all of the Ampr and Chlr vectors(no unexpected restction sites).<br />
<br/><br />
<br/><br />
<p><strong>Testing method</strong><br />
<span></span>All the sRNA generators with artificial region were tested with inducer gradient, to verify <i>spot42</i> of different levels repression efficiency in vivo.(<a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">Results</a>)<br />
<br/><span></span>We tested Comparators ligated with <i>srlA</i>/<i>ytfJ</i>/<i>nanC</i> buffer RNA with aTc/IPTG 2-dimension gradient using well designed <a href="https://2012.igem.org/Team:OUC-China/protocol">protocol</a>,the data is given in <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result">results</a> page,while the data of ratio sensor is still in progress.<br />
</br><br />
</br><br />
<p><strong>Adjustment</strong><br />
<span></span>We also made some adjustment on the mRNA synthesis rate(αm) by replacing our device to different copy number vectors.We assemblied two sRNA generator together to J61002(Ampr) while ligating GFPmRNA_Generator to pSB4C5(Chlr), to perform double-plasmids transformation.But the function of buffer RNA didn’t work well due to unclear reasons.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:5px;" src="https://static.igem.org/mediawiki/2012/d/d7/Ouc-project-designmaking-design19.jpg"/></a><br />
<br/><br />
<br/><br />
<p><strong>Note</strong><br />
<span></span>1/ColE1 is a medium copy number replication origin(~70 copies per cell),whereas p15A is a low one(~5 copies per cell).<br />
<br/><span></span>2/The efficiency of endogenous terminator of <i>spot42</i> remains unclear. Another consideration of swiching vectors is to avoid the readthrough of <i>spot42</i>.<br />
<br/><span></span>Some evidence suggests it’s not very strong: when induces <i>spot42</i> in the upstream of GFP_Generator with very high aTc concentration, the GFP level soared to a very high level(even higher than GFP_generator alone), made it elusive on this paradox.<br />
<br/><br />
<br/><br />
<p><strong>Project flow chart</strong><br />
<br/><br />
<br/><br />
<a><img style="margin-left:35px;" src="https://static.igem.org/mediawiki/2012/f/f3/Ouc-project-designmaking-design20.JPG" /></a><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Safety
Team:OUC-China/Safety
2012-10-27T02:13:41Z
<p>PengYong: </p>
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<h1 style="border:none;">Safety Questions</h1><br />
<p><strong>1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?</strong><br />
<p><span></span>First of all, the safety of the researchers is guaranteed. In the molecular laboratory, many chemicals such as EB pose a threat to human beings, so we use chemicals with higher safety coefficients instead. For example, EB can be replaced by Gel Green and Gel Red. At the same time, we place and deal with the toxic chemicals in an isolated area in case we pollute other chemicals or laboratory items. Besides, we assign one person to do safety-checks every morning (before 8:00a.m.), every afternoon (12:00a.m.~1:30p.m.) and every night(after10:00p.m.) to reduce the possibility of safety problems.</p><br />
<p><span></span>Secondly, we insure public safety when we do experiments. Because many chemicals are toxic and many bacteria are resistant to antibiotics, chemicals and bacteria are taken in and out of the laboratory carefully. Apart from garbage or necessary things, everything taken into laboratory can’t be taken out in order to prevent toxic substances and transgenic bacteria from coming out of the laboratory.</p><br />
<p><span></span>Lastly, environmental safety is also considered. Before being discarded, all transgenic bacteria grown on the culture mediums is sterilized by autoclave so that transgenic bacteria won’t spread. Toxic garbage is dealt with carefully. All garbage is placed individually and recycled by a special department. There is someone who is responsible for storing and using antibiotics when they are needed and making sure that antibiotics aren’t taken out of the laboratory.</p><br />
<br/><br />
<p><strong>2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</strong></p><br />
<p><span></span>All the frameworks of plasmids we use in iGEM are from MIT parts registry. Because there are antibiotic resistance genes on the plasmids, bacteria carrying the plasmids may recombine genes with other bacteria. This can result in bacteria with resistance to many antibiotics, so we pay special attention to transgenic bacteria. The transgenic bacteria to be eliminated need to be sterilized by autoclave, and the garbage is dealt with alone.</p><br />
<p><span></span>The strains we use in this project are Planktothrix rubescens and Escherichia coli. In the studies about Synthetic Biology, Cyanobacteria and E. coli have been widely used. Some commercial strains, such as Top 10 strain, have been proved harmless to the human body. Although many species of Cyanobacteria and E. coli are toxic to human beings, the strains we chose are nontoxic. All the experiments are done in the laboratory, and the bacteria can’t be taken out of the laboratory. Our work will be able to decrease the possibility of biosafety problems of the public and the environment.</p><br />
<br/><br />
<p><strong>3. Is there a local biosafety group, committee, or review board at your institution?</strong></p><br />
<p><span></span>As for biosafety and laboratorial safety, our college has been formulated according to laboratory safety management regulations-laboratory safety system, which is aimed at our iGEM laboratory, iGEM team member experiment rules and laboratory management rules. In order to promote safety education in college, under the promotion of the students, the school has set up the compulsory course "experiment equipment use procedures and laboratory safety" for all freshmen. At the same time, our laboratory has established a complete safety management system, designed to ensure safety during the project progress in the laboratory, and to eliminate hidden dangers of the lab. As for waste in the experiment, our college has a special waste recycling mechanism to avoid the outflow of hazardous waste. Besides, the People’s Republic of China issued a document “Laboratories—General requirements for biosafety” (GB19489-2004) on April the 5th 2004. It can be viewed at <a href="http://www.iphy.ac.cn/aqzt/aqzt_xzzx/201105/P020110503609211780348.pdf"> here</a>!</p><br />
<p><span></span>To further strengthen our laboratory safety management, we had been dedicated to Qingdao institution of Bioenergy and Bioprocess Technology Chinese Academic of Sciences professors The professors provided advice about the safety management of laboratory system, hoping to make improvements on our own safety management mechanism based on their advice and practice system. Also, we hope that the experts of the institution will make an assessment on the safety of the experimental project and make suggestions to ensure the safety of the biolab.</p><br />
<p><span></span> In order to let more members be aware of the importance of biological safety and lab safety, we are going to set up our own community about promoting biological safety and laboratory safety. We hope to arouse attention to biological safety and laboratory safety in every laboratory in our college.</p><br />
<br/><br />
<p><strong>4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</strong></p><br />
<p><span></span>Before our team took part in iGEM , our teachers educated us on the safety aspects of experiments, and all the members have learned about the safe use of instruments in order to proceed the experiment safely. We recommend that each laboratory write its own laboratory safety management manual. Our members do a safety check daily as special duty laboratory technicians. The laboratory also arranges a security officer to check the instruments and to manage the security of the laboratory.</p><br />
<p><span></span>In order to ensure that genetically modified bacteria from the lab cannot survive in places where it shouldn’t, we suggest that the bacteria used in the experiment be nutritionally deficient and be developed in nutrition supplemented culture medium. As a result, leaked bacteria would die of nutrition deficiency. For instance, 2011 OUC-China iGEM team used auxotroph bacteria to achieve a multiple symbiotic system. The effect is that one colony is not able to live independently in the in the system of symbiosis. <a href="https://2011.igem.org/Team:OUC-China">(https://2011.igem.org/Team:OUC-China)</a></p><br />
<p><span></span>As for the selection of strains, we suggest that each iGEM team replace pathogenic strains with non-pathogenic strains. Bacteria implanted with toxins genes can be added with LVA tags to speed up toxin protein degradation. </p><br />
<p><span></span>In addition, we also suggest that some kind of characteristic sequence be added to standard plasmids, to make standard plasmid DNA easy to identify through DNA barcode technology. In this way we can determine whether or not a certain kind of bacteria has gone under standard plasmid reconstruction.</p><br />
<p><strong>5. How to prevent the floating bacteria from turning into ecological disaster?</strong><br />
<p><span></span>To prevent our floating biosensor from propagating to be a ecological disaster in ocean,here we adopt cell lysis system coupled with quorum sensing system as our cell density restrainer. <br />
<br/><br />
When cell density overweighs a fixed value in biosensor, the self-lysis system will be induced by quorum-sensing signals such as AHL, initiating cell death program to restrain the propagation.<br />
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<h1 style="border:none;">Safety Questions</h1><br />
<p><strong>1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?</strong><br />
<p><span></span>First of all, the safety of the researchers is guaranteed. In the molecular laboratory, many chemicals such as EB pose a threat to human beings, so we use chemicals with higher safety coefficients instead. For example, EB can be replaced by Gel Green and Gel Red. At the same time, we place and deal with the toxic chemicals in an isolated area in case we pollute other chemicals or laboratory items. Besides, we assign one person to do safety-checks every morning (before 8:00a.m.), every afternoon (12:00a.m.~1:30p.m.) and every night(after10:00p.m.) to reduce the possibility of safety problems.</p><br />
<p><span></span>Secondly, we insure public safety when we do experiments. Because many chemicals are toxic and many bacteria are resistant to antibiotics, chemicals and bacteria are taken in and out of the laboratory carefully. Apart from garbage or necessary things, everything taken into laboratory can’t be taken out in order to prevent toxic substances and transgenic bacteria from coming out of the laboratory.</p><br />
<p><span></span>Lastly, environmental safety is also considered. Before being discarded, all transgenic bacteria grown on the culture mediums is sterilized by autoclave so that transgenic bacteria won’t spread. Toxic garbage is dealt with carefully. All garbage is placed individually and recycled by a special department. There is someone who is responsible for storing and using antibiotics when they are needed and making sure that antibiotics aren’t taken out of the laboratory.</p><br />
<br/><br />
<p><strong>2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</strong></p><br />
<p><span></span>All the frameworks of plasmids we use in iGEM are from MIT parts registry. Because there are antibiotic resistance genes on the plasmids, bacteria carrying the plasmids may recombine genes with other bacteria. This can result in bacteria with resistance to many antibiotics, so we pay special attention to transgenic bacteria. The transgenic bacteria to be eliminated need to be sterilized by autoclave, and the garbage is dealt with alone.</p><br />
<p><span></span>The strains we use in this project are Planktothrix rubescens and Escherichia coli. In the studies about Synthetic Biology, Cyanobacteria and E. coli have been widely used. Some commercial strains, such as Top 10 strain, have been proved harmless to the human body. Although many species of Cyanobacteria and E. coli are toxic to human beings, the strains we chose are nontoxic. All the experiments are done in the laboratory, and the bacteria can’t be taken out of the laboratory. Our work will be able to decrease the possibility of biosafety problems of the public and the environment.</p><br />
<br/><br />
<p><strong>3. Is there a local biosafety group, committee, or review board at your institution?</strong></p><br />
<p><span></span>As for biosafety and laboratorial safety, our college has been formulated according to laboratory safety management regulations-laboratory safety system, which is aimed at our iGEM laboratory, iGEM team member experiment rules and laboratory management rules. In order to promote safety education in college, under the promotion of the students, the school has set up the compulsory course "experiment equipment use procedures and laboratory safety" for all freshmen. At the same time, our laboratory has established a complete safety management system, designed to ensure safety during the project progress in the laboratory, and to eliminate hidden dangers of the lab. As for waste in the experiment, our college has a special waste recycling mechanism to avoid the outflow of hazardous waste. Besides, the People’s Republic of China issued a document “Laboratories—General requirements for biosafety” (GB19489-2004) on April the 5th 2004. It can be viewed at <a href="http://www.iphy.ac.cn/aqzt/aqzt_xzzx/201105/P020110503609211780348.pdf"> here</a>!</p><br />
<p><span></span>To further strengthen our laboratory safety management, we had been dedicated to Qingdao institution of Bioenergy and Bioprocess Technology Chinese Academic of Sciences professors The professors provided advice about the safety management of laboratory system, hoping to make improvements on our own safety management mechanism based on their advice and practice system. Also, we hope that the experts of the institution will make an assessment on the safety of the experimental project and make suggestions to ensure the safety of the biolab.</p><br />
<p><span></span> In order to let more members be aware of the importance of biological safety and lab safety, we are going to set up our own community about promoting biological safety and laboratory safety. We hope to arouse attention to biological safety and laboratory safety in every laboratory in our college.</p><br />
<br/><br />
<p><strong>4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</strong></p><br />
<p><span></span>Before our team took part in iGEM , our teachers educated us on the safety aspects of experiments, and all the members have learned about the safe use of instruments in order to proceed the experiment safely. We recommend that each laboratory write its own laboratory safety management manual. Our members do a safety check daily as special duty laboratory technicians. The laboratory also arranges a security officer to check the instruments and to manage the security of the laboratory.</p><br />
<p><span></span>In order to ensure that genetically modified bacteria from the lab cannot survive in places where it shouldn’t, we suggest that the bacteria used in the experiment be nutritionally deficient and be developed in nutrition supplemented culture medium. As a result, leaked bacteria would die of nutrition deficiency. For instance, 2011 OUC-China iGEM team used auxotroph bacteria to achieve a multiple symbiotic system. The effect is that one colony is not able to live independently in the in the system of symbiosis. <a href="https://2011.igem.org/Team:OUC-China">(https://2011.igem.org/Team:OUC-China)</a></p><br />
<p><span></span>As for the selection of strains, we suggest that each iGEM team replace pathogenic strains with non-pathogenic strains. Bacteria implanted with toxins genes can be added with LVA tags to speed up toxin protein degradation. </p><br />
<p><span></span>In addition, we also suggest that some kind of characteristic sequence be added to standard plasmids, to make standard plasmid DNA easy to identify through DNA barcode technology. In this way we can determine whether or not a certain kind of bacteria has gone under standard plasmid reconstruction.</p><br />
<p><strong>5. How to prevent the floating bacteria from turning into ecological disaster?</strong><br />
<p><span></span>To prevent our floating biosensor from propagating to be a ecological disaster in ocean,here we adopt cell lysis system coupled with quorum sensing system as our cell density restrainer. <br />
<br/><br />
When cell density overweighs a fixed value in biosensor, the self-lysis system will be induced by quorum-sensing signals such as AHL, initiating cell death program to restrain the propagation.<br />
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Team:OUC-China/Parts
2012-10-27T02:04:39Z
<p>PengYong: </p>
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<h1 style="border:none;">Summary</h1><br />
<p><strong>Parts submitted:</strong></p> <p>33 Composite expression modules</p> <br />
<p>10 Generator</p><br />
<p>4 Regulatory promoters</p><br />
<p> 1 Ribosome Binding Sites</p><br />
<p> 10 Coding proteins</p><br />
<p> 10 RNA </p> <br />
<p>Total: <strong>68 Parts</strong></p> <br />
<br />
<p>All 68 parts have been submitted to the registry and can be found <a <br />
<br />
href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2012&group=OUC-China">here</a>.</p><br />
<br />
<br />
<br/><br />
<h1 style="border:none;">Data for favorite parts</h1><br />
<br />
<p><strong>Gvp cluster(<a href="http://partsregistry.org/Part:BBa_K737010">BBa_K737010</a>)</strong> is a 1 kbp part <br />
<br />
contains 2 open reading frames coding for gas vesicle genes (gvpA gvpC-20) from Bacillus Planktothrix rubescens strain BC-<br />
<br />
Pla 9402. Promotion of the sequence results in expression of gas vesicles, organelles made entirely out of protein. These <br />
<br />
organelles contain gas and therefore provide bouyancy to the cell. This slows the rate of settling of the cells in <br />
<br />
different water layer from Lake Zurich by gas vesicles, see Walsby 2002.</p><br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/d/d5/Ouc-parts1.jpg"/></div><br />
<br />
<p><span>the expression situation of the Gas vesicle gene cluster in Escherichia coli JM109, from the picture, we can see <br />
<br />
obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon. The picture on the right of <br />
<br />
the experimental group were liquid surface layer and intermediate layer of fluid sampling results, we can see that the <br />
<br />
fluid layer surface bacteria volume density is obviously higher than that of the middle layer of fluid.</span></p><br />
<br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/b/bf/Ouc-parts2.png"/></div><br />
<br />
<br />
<p><span>The picture shows the gas vesicle in the cells after expressed in the overall trend. We in the cultivation of the <br />
<br />
cells into logarithmic phase after transferring them to test tube, quiet place and culture 36 hours after using Trace <br />
<br />
stratified take liquid apparatus at the same time to each liquid layer for sampling. Then each layer of fluid samples of <br />
<br />
blood count the number of cells with statistics. Results show that gas vesicle due to the existence of the cell <br />
<br />
concentration in the test tube surface layer of fluid, and control group due to the action of gravity that most cells <br />
<br />
sedimentation in the test tube bottom.</span></p><br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/4/4c/Ouc-parts3.png"/></div><br />
<p><span>Graph of OD measurements at 600nm of both experience group and control group. From the experimental results can be <br />
<br />
seen in the liquid surface .There cell number have significant abrupt transformation.</span></p><br />
<br/><br />
<br />
<p><strong>Constitutive GVPA Generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737006">BBa_K737006</a>) It contains a constitutive promoter, GvpA protein , GFP protein and a <br />
<br />
terminator.This part can be used as a control of (J23106+P0412+R0011+B0034+GVPA+E0840). It can express GvpA protein and GFP <br />
<br />
constantly. When it transformed with (J23106+P0440+R0040+B0034+GVPC+E0840), the bacteria may can float with GFP!</p><br />
<br />
<p><strong>YtfJ_Comparator</strong>( <a href="http://partsregistry.org/Part:BBa_K737045">BBa_K737045</a>) The leading <br />
<br />
sequence involved in the YtfJ transcript which can act as the target of the sRNA, spot42. It enables strong competition <br />
<br />
with the galk::GFP to interact with spot42 which shows great potential for constructing sRNA-mediated circuit.</p><br />
<p>This device mean to test feasibility of N/P comparator,replacing Nitrate,Phosphate inputs by IPTG,aTc。Small RNA Spot42 <br />
<br />
controls the GFP mRNA translation by blocking the RBS in galK.The galK and E0040 is fused together using ClaI site to avoid <br />
<br />
stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.</p><br />
<br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/9/97/Ouc-parts4.png"/></div><br />
<br />
<p>Spot42 sRNA is under control of aTc inducible device,the buffer RNA YtfJ is under control of IPTG inducible device.</p><br />
<br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/a/a4/Ouc-parts5.jpg"/></div><br />
<br />
<p><strong>Constitutive GVPC Generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737007">BBa_K737007</a>)It contains a constitutive promoter, GvpC <br />
<br />
protein , GFP protein and a terminator.This part can be used as a control of (J23106+P0440+R0040+B0034+GVPC+E0840). It can <br />
<br />
express GvpC protein and GFP constantly. When it transformed with (J23106+P0412+R0011+B0034+GVPA+E0840), the bacteria may <br />
<br />
can float with GFP!</p><br />
<p>Here is the result of SDS-polyacrylamide gel electrophoresis. We can see GFP and GvpC protein directly. Lane 4 is top10 <br />
<br />
control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper <br />
<br />
white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is <br />
<br />
GvpC-20 (20KD). Because the MW(molecular weight) of GvpA protein is too small, it’s very difficult to see it on the SDS-<br />
<br />
polyacrylamide gel. But the expression of GFP suggests it may be expressed.<br />
Some papers say that GvpA is difficult to see is that GvpA is too hydrophobic to run out of sample hole.</p><br />
<br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/8/88/Ouc-parts6.jpg"/></div> <br />
<p><span>Figure1.The result of SDS-polyacrylamide gel electrophoresis. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper white boxes are in the position about 26KD are GFP protein (25.8KD). The lower white box in the position of about 20KD is GvpC-20 (20KD).</span></p><br />
<p>Because the GvpA protein can’t be seen on the SDS-polyacrylamide gel electrophoresis, we wanted to use GFP to detect the expression of GvpA. We use a polycistron of gvpA(gvpC) and gfp to detect the RFU. We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control. First, the RFU of these parts are rather high. Second, the E.Coli with these two plasmids can float. Third, we can see GvpC on the SDS-polyacrylamide gel. So, we can see the two parts can express Gvp Protein and work well.</p><br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/8/80/Ouc-parts7.jpg"/></div> <br />
<p></span>Figure2.We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control.</span></p><br />
<p><strong>SrlA_Comparator</strong>(<a href="http://partsregistry.org/Part:BBa_K737046">BBa_K737046</a>)The leading sequence involved in the SrlA transcript which can act as the target of the sRNA, spot42. It enables strong competition with the galk::GFP to interact with spot42 which shows great potential for constructing sRNA-mediated circuit.<br />
This device mean to test feasibility of N/P comparator,replacing Nitrate,Phosphate inputs by IPTG,aTc.</p><br />
<p>Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK.The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.</p><br />
<br />
<p>Spot42 sRNA is under control of aTc inducible device,the buffer RNA SrlA is under control of IPTG inducible device.</p><br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/0/02/Ouc-parts8.jpg"/></div><br />
<p><strong>Spot42</strong> (<a href="http://partsregistry.org/Part:BBa_K737058">BBa_K737058</a>)is a multitarget small RNA that mediates the discoordinate expression of the E.coli galactose operon and other sugar metabolic pathway,such as galK,nanC,ytfJ,srlA,which are the 5’ leader sequence of the corresponding metabolic enzymes.<br />
<div class="image"><img src="https://static.igem.org/mediawiki/2012/1/1f/Ouc-parts9.jpg"/></div> <br />
<p>Take galK for example,Spot42 causes translation repression by base pairing to RBS in the 5’ leader sequence,then block the recognition of the antiSD sequence on 30S subunit.<br />
It’s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo¨ rg Vogel,for it doesn’t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.Spot42-galK complex degrades in a slow and presently unclear way. The base pairing is initiated by the recognition of seed region,which plays a significant role in the repression efficiency.</p><br />
<br />
<p>Spot42 have a weak Hfq binding site(whereas galK weaker),an endogenous terminator(unclear efficiency,maybe weak),and the multitarget repression stem-loop.</p><br />
<br/><br />
<h1 style="border:none;">Data for optimized parts</h1><br />
<p><strong>galK:GFP generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737034">BBa_K737034</a>) Compared with the original part, our new part possesses<br />
new function.Small RNA Spot42 controls the GFP mRNA translation by blocking the RBS in galK. The galK and E0040 is fused together using ClaI site to avoid stop condon in standard scar.J23106 is a moderate promotor,and galK’s RBS is a moderate one.</p><br />
<br />
<p><strong>GFP generator</strong>(<a href="http://partsregistry.org/Part:BBa_K737068">BBa_K737068</a>) is provided by MIT in 2012 plates for teams. But we couldn’t transfer it successfully. So we constructed it again and submitted it to registry.</p><br />
<br />
<p><strong>OmpR</strong>(<a href="http://partsregistry.org/Part:BBa_K737067">BBa_K737067</a>) Positively regulated, OmpR-controlled promoter ligated with B0034 can be used to control genetic circuit by light with light-dependent control element. This promoter is taken from the upstream region of OmpC. Phosphorylated OmpR binds to the three operator sites and activates transcription.</p><br />
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PengYong
http://2012.igem.org/Team:OUC-China/HumanPractice/SpecialHM
Team:OUC-China/HumanPractice/SpecialHM
2012-10-27T02:02:26Z
<p>PengYong: </p>
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<h1 style="border:none;">Special OUC-China HP</h1><br />
<h2>1.Relay in OUC-China</h2><br />
<p><span></span>We are not just a team.<br />
<br/><span></span>OUC-China iGEM is not just a team. Up to now, we have three teams—OUC-China 2011, 2012 and 2013. We are not like other teams, we have relay. When busy in preparing 2011 iGEM jamboree, 2011 OUC iGEMers took their time to teach 2012 iGEMers molecular biology and introduce synthetic biology to them. To educate 2012 members better, they used every minute to learn and taught more. Some of their team even helped us and taught us experiment after they graduated.<br />
<br/><span></span>To the same goal, this year, we taught biochemistry and microbiology to freshman in our university. In addition, to make iGEM and synthetic biology known to them, we held lectures (about iGEM ), meetings (with Peaking University)and camps (with Qingdao No.2 High School and learn synthetic biology).<br />
<br/><span></span>Our team is not just a team. It’s a home where we can treat as brothers and sisters. No matter what medal we got, we would appreciate OUC-China 2011 iGEM team and hope OUC-China 2013 iGEM do a better job!<br />
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<h2>2.Respect nature</h2><br />
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<p style="margin-left:380px; margin-top:-340px;"><span></span>Human beings have been making significant changes to the face of the earth for thousands of years. We have made buildings and dug tunnels; we have sent people to the moon and reached the bottom of the sea. We have also changed the lives of many of our fellow organisms. To what extent can we tolerate our actions that are creating an increasing impact on our earth? Is it a violation of nature's laws what biologists nowadays are doing?<br />
<br/><span></span>Different people have different views on what humans are "allowed" to do. One example of radical views may be the fruitarian.Fruitarians only eat fruits and nuts that have fallen from its branches naturally, trying not to make any impact on other </p><p>living organisms. We believe that this is wrong and unnatural, as humans are consumers designed to live on other organisms so it is unavoidable that we make an impression on our world. The other extreme may be the biologist who changes the genetic material of life, transforming their role in ecology, perhaps even altering the course of evolution. The question is to what extent can humans accept their own actions. <br />
<br/><span></span>In our opinion, every form of life has its impact on the environment and other living things. Humans' reform on nature has always been one part of its construction. The earth has undergone many changes in its long history, each change ending up reaching a new ecological balance. Synthetic biology is opening up new opportunities and bringing about new changes. This is a natural progress.<br />
<br/><span></span> Most may say that humans possess more ability to change the world than any other creature that has existed on the earth. Greater ability brings forth the need for further responsibility. We must be aware of what we are doing and avoid altering life into an undesirable form. For example, our project is aimed at preventing pollution and protecting the marine environment. This is what we scientists should be doing: making a positive impact on the earth.<br />
</p><br />
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<h2>3.Environmental survey</h2><br />
<p><span></span> Because our project of this focuses on the ratio of N and P and cyanobacteria in the sea water, we consulted professors who study on ecology to get more information about it. Our university is located in Qingdao, Shandong Province, near the Kiaochow Bay, so we made a environmental survey there to collect phytoplankton there and learned how to classify them. In particular, we made this survey with freshmen in our university during 2th summer camp of life science and technology. We think we can learn more about red tide and cyanobacteria from this survey. We let more students to join the survey and learn with us in this survey. It not only made us better, also was a good chance to introduce iGEM, our project, and most importantly, conception of protecting the environment to them. It’s a new approach to held a survey in camp to introduce iGEM and do human practice, we hope it can help other teams.</p><br />
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PengYong
http://2012.igem.org/Team:OUC-China/HumanPractice/Camps
Team:OUC-China/HumanPractice/Camps
2012-10-27T01:58:14Z
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<h1 style="border:none;">Camps, Class and Lectures</h1><br />
<h2>1.Hands-on experience summer camp with Qingdao No.2 High School.</h2><br />
<p>This summer, we held a camp with 28 high school students in our university. Our goal was to motivate their interest of bioscience by doing experiments and teach them how to learn by oneself (Internet is a good choice). In this camp, we prepared many experiments about molecular biology, such as miniprep and PCR.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*Why we held this meeting?</p><br />
<p><span></span>We want to let more people know about synthetic biology and pay attention to biosafety. Now, synthetic biology still is a developing subject in China. Many students even haven’t heard about it. As the hard core of developing bioscience and the future of the world, the more knowledge they get, the better the world is. Besides, by sharing our knowledge with them, we trained our skills of talking and understood the concept of synthetic biology with deeper expression.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*What we did in this camp?</p><br />
<p><span></span> In this camp, we gave many classes to participants, such as the principle of PCR, introduction of synthetic biology and iGEM. During the class, we shared the concept of iGEM and standard and let more students have interests on synthetic biology. In addition to classes, we also teach them to do molecular experiments, such as miniprep and PCR. By this means, we trained our experiment and communication skills. We also stressed the importance of safety, including researcher safety, public safety, or environmental safety.<br />
At the end of this camp, we gave out a questionnaire and analyzed their answers. <br />
<br/><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/0/03/Ouc-human-practice-kangincamps0.jpg" /><br />
<img style="margin-left:380px;margin-top:-270px;" src="https://static.igem.org/mediawiki/2012/a/a4/Ouc-human-practice-kangincamps.jpg"/></a><br />
<p style="margin-left:80px;">Teaching the principle of PCR<sub></sub><span></span><span></span><span></span>Teaching the experiment of miniprep</p><br />
<br/><br />
<p>Here are the questionnaires and analysis:<br />
<br/><br />
<br/><br />
<h2>2.2th summer camp of life science and technology</h2><br />
<p style="text-decoration:underline;">*Why we held this camp?</p><br />
<p><span></span> Last year, we held 1st summer camp of life science and technology. Through this camp, most of the participants (many of them are OUC iGEMers) had developed their interests in synthetic biology and iGEM. After that, we studied molecular biology and introduction to synthetic biology by ourselves. This year, we become OUC-China 2012 iGEMers.<br />
<br/><span></span>So, aiming at educating new iGEMers for OUC-China, we introduced synthetic biology and iGEM during this camp. Besides, hoping they can appreciate the beauty of nature and learn more far from books, we made a sea investigation in this camp. We love synthetic biology because we love where we live!<br />
<br/><br />
<br/><br />
<a href="https://static.igem.org/mediawiki/2012/9/93/One_of_freshmen%27s_posters.zip"><img src="https://static.igem.org/mediawiki/2012/f/f0/Ouc-human-practice-poster.jpg" /></a><br />
<p style="margin-left:70px;">A poster of students in this camp</p><br />
<br />
<p style="margin-left:370px; margin-top: -260px; text-decoration:underline;">*What did we do during this camp?</p><br />
<a><img style="margin-left:370px; margin-top:-310px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-human-practice-seainvestigation.jpg" /></a><br />
<p style="margin-left:370px;"> After one year in university, most participants had learned botany and zoology. To build the general system of biology, we taught them biochemistry and did some microbiology experiments. Now, biology has many subjects and many new disciplines emerge endlessly. To make more new disciplines known to them, we held a lecture during this camp to introduce bioinformatics, system biology and of course, synthetic biology.<br />
</p><br />
<br/><br />
<br/><br />
<h2>3.Winter camp for brain storming</h2><br />
<p style="text-decoration:underline;">*Brain storming in winter vacation with professors</p><br />
<p><span></span> During winter vacation, we went back school early to brain storm. During the ten days’ camp, every iGEMers in our team read papers, speak out what they leant in winter vacation, talked about ideas and communicate with professors together. In our lab, we were enjoying the joy of science and exploration.</p><br />
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<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/4/49/Ouc-human-practice-meeting.jpg" /></a><br />
<p style="text-align:center;">Brainstorming with professors</p><br />
<br/><br />
<br/><br />
<p><span></span>Living and working with each other for ten days was an inspiring experience for 2012 OUC-iGEMers. This was the first time for our team to get the feeling of being a true researcher and to use our own imagination to create things that we have never dreamed of before. Being fully absorbed in expressing our ideas with the most terrific teammates is something that a young person just has to experience!</p><br />
<h2>4.Popularization of bioscience with young kids</h2><br />
<p><span></span>In June, participants from the 2012 iGEM teams joined a multitude of presentations to expand the knowledge of No.39 middle school’s students in the general sciences. Presentations included marine biology, bioengineering, introduction to biology and so on. Besides we prepared various interactive sessions for students to learn and practice experimental skills. We also displayed our work on the ‘Wu Xing’ system in 2011 iGEM and ratio sensor in this summer. Through this process, students could be exposed to recent scientific research.<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/6/65/Ouc-human-practice-gonginteaching.jpg"></a><br />
<p style="text-align:center;">Minghao Gong is communicating with young kids</p><br />
<br/><br />
<br/><br />
<h2>5.Introduce our project to professors and students in our university.</h2><br />
<p><span></span>During winter vacation, we had a brainstorming and our idea was settled during April. To make iGEM and our project known in our university, we made a presentation to introduce our project to who are interested in our project, including professors and students.<br />
<br/><span></span>Last year, OUC-China iGEM Team got a bronze medal in Asia Jamboree. It attracted many students to learn about synthetic biology and iGEM. Many students are curious about synthetic biology and its concept. As iGEMers, we wanted to use an example to explain iGEM and synthetic biology. Since we have our own idea and needed a test, we made a presentation to introduce iGEM and our idea to others. <br />
<br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/2/28/Ouc-human-practice-toothers.jpg" /></a><br />
<p style="text-align:center;">Describe our project to others</p><br />
<br/><br />
<br/><br />
<h2>Lecture of iGEM in OUC</h2><br />
<p><span></span>In April, we have the honor to meet with Haoqian Zhang and Shuobing Chen, the former and current iGEM team leader of Peking University respectively. We displayed multitude of brainstormed ideas in the last winter vacation and make a deep discussion with them. Through this process, we further understood the core thinking pattern and application of synthetic biology. They are very kind and ready to help us along with the coming 2012 iGEM competition. Here we express our sincere appreciations to Peking iGEM team. Finally, we invited them to perform a presentation named ‘research-driven education’ which embodied their brand-new concepts on the relation between education and scientific research. Besides, they displayed their team’s work from 2007-2011 and the joy and sorrow along the inheriting process. We not only expanded our knowledge from their lectures, but deeply recognized our responsibility to inherit and spread the edge-cutting knowledge as well as concepts to those ready to achieve their goals in scientific research. We’re ready to assume the responsibility to impel the education reform. Are you ready?<br />
<br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/a/a7/Ouc-human-practice-afterlecture.jpg" /></a><br />
<p style="text-align:center">A group photo after this lecture</p><br />
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PengYong
http://2012.igem.org/Team:OUC-China/HumanPractice/Communication
Team:OUC-China/HumanPractice/Communication
2012-10-27T01:56:52Z
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<h1 style="border:none;">Meeting and Academic Communication</h1><br />
<h2>1/Meeting with Peking iGEM Team</h2><br />
<p> As a new born iGEM team,questions below puzzled us for a very long time.<br />
However,Peking Univerisity iGEM team helped us a lot by giving us directions willingly.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*How about biosafety issues?</p><br />
<p><span></span>“Biosafety is not a far-reaching issue for us,in fact,”Said Robin Chen,”biological weapons is within reach by means of synthetic biology.The reproducible nature of life will be the most horrible aspect as a weapon.”<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*What’s our role in the synbio globalization progress?</p><br />
<p><span></span> It’s really a good idea to ultilize the creativity and passion of youngmen all over the world to explore new ideas.”It is like a high throughput machine, putting many novel ideas into practice without paying a single dollar.”Said Zhang Peiran,a modeller of OUC.<br />
It is really a brillant idea,after all.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*What the item synthetic biology actually mean?</p><br />
<p><span></span>“Synthetic biology is based on previous works of molecular biology actually,under the guidance of system biology,quantitatively understand and construct a part,a device,even a system.”Said Zhang Haoqian,who had participated in iGEM as long as four years,now the leader of Institue of Synthetic and System Biology in Peking University,”What it(synbio) matters is that the part or device you made on your project really works.”<br />
<br/><br />
<br/><br />
<br />
<a><img src="https://static.igem.org/mediawiki/2012/9/99/Ouc-human-practice-Academic-Communication-in-Peaking-University.jpg"/></a><br />
<br/><br />
<br/><br />
<h2>2/A Visit to Institute of Microbiology Chinese Academy of Science</h2><br />
<p style="text-decoration:underline;">*What’s the relationship bewteen microbiology and synthetic biology?</p><br />
<p><span></span>2011 Winter, we visited Institure of Microbiology CAS,which made us more clear on the relationship between Synbio and Microbiology.We realized that microbes are the most wonderful things in the world in terms of Synbio,microbes are a wonderful gene library for deriving biobricks.And, we can see the enormous protential of other species other than E.coli in energy,health,enviroment…</p><br />
<br/><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/03/Ouc-human-practiceIMCAS.jpg"/></a><br />
<br/><br />
<br/><br />
<h2>3/iGEM in QIBEBT</h2><br />
<p style="text-decoration:underline;">*What is QIBEBT?</p><br />
<p><span></span>QIBEBT is short for Qingdao Institute of Bioenergy and Bioprocess Technology.Aiming at producing renewable biofuels by plants,QIBEBT utilize synthetic biology approach to engineer a series of energy plants like fuel algae.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*Researcher Xu Jian Visited Ouc iGEM Lab</p><br />
<p><span></span> Last year,our instructor,researcher Xu Jian visted our lab and shared his comments on synthetic biology with us,then we reached an agreement of cooperation.<br />
<br/><br />
<br/><br />
<a style="margin-left:70px;"><img src="https://static.igem.org/mediawiki/2012/e/ea/Ouc-human-practice-xujian.jpg"/></a><br />
<br/><br />
<p style="text-align:center;">Our Instructor Researcher Xu Jian Visited Ouc iGEM Lab</p><br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*Making iGEM known in QIBEBT</p><br />
<p><span></span>“The question that fresh labmen come up with always concerns with the most basic problem,”says Ma Bo,another instructor of us,”and they always unscarred by experimental depression or old sterotypes,that is the most invaluable idea for our lab.”<br />
<br/><br />
<br/><br />
<a style="margin-left:180px;"><img src="https://static.igem.org/mediawiki/2012/4/4d/Ouc-human-practice-gonginimcas.jpg"/></a><br />
<p style="text-align:center;">Performing Experiments in QIBEBT</p><br />
<br/><br />
<p><span></span>Through we performed most of our experiments in QIBEBT,which focused on synthetic biology engineering,little knowed iGEM including our instructor,they even thought we were playing house like child!We were depressed and determined to change their attitude,to this end, we showed our enthusiasm and sharpness as an iGEMer,many researchers were impressed by the novel idea of iGEM projects,rigorous experimental design and detailed datasheet!<br />
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PengYong
http://2012.igem.org/Team:OUC-China/HumanPractice/Communication
Team:OUC-China/HumanPractice/Communication
2012-10-27T01:56:30Z
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<h1 style="border:none;">Meeting and Academic Communication</h1><br />
<h2>1/Meeting with Peking iGEM Team</h2><br />
<p> As a new born iGEM team,questions below puzzled us for a very long time.<br />
However,Peking Univerisity iGEM team helped us a lot by giving us directions willingly.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*How about biosafety issues?</p><br />
<p><span></span>“Biosafety is not a far-reaching issue for us,in fact,”Said Robin Chen,”biological weapons is within reach by means of synthetic biology.The reproducible nature of life will be the most horrible aspect as a weapon.”<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*What’s our role in the synbio globalization progress?</p><br />
<p><span></span> It’s really a good idea to ultilize the creativity and passion of youngmen all over the world to explore new ideas.”It is like a high throughput machine, putting many novel ideas into practice without paying a single dollar.”Said Zhang Peiran,a modeller of OUC.<br />
It is really a brillant idea,after all.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*What the item synthetic biology actually mean?</p><br />
<p><span></span>“Synthetic biology is based on previous works of molecular biology actually,under the guidance of system biology,quantitatively understand and construct a part,a device,even a system.”Said Zhang Haoqian,who had participated in iGEM as long as four years,now the leader of Institue of Synthetic and System Biology in Peking University,”What it(synbio) matters is that the part or device you made on your project really works.”<br />
<br/><br />
<br/><br />
<br />
<a><img src="https://static.igem.org/mediawiki/2012/9/99/Ouc-human-practice-Academic-Communication-in-Peaking-University.jpg"/></a><br />
<br/><br />
<br/><br />
<h2>2/A Visit to Institute of Microbiology Chinese Academy of Science</h2><br />
<p style="text-decoration:underline;">*What’s the relationship bewteen microbiology and synthetic biology?</p><br />
<p><span></span>2011 Winter, we visited Institure of Microbiology CAS,which made us more clear on the relationship between Synbio and Microbiology.We realized that microbes are the most wonderful things in the world in terms of Synbio,microbes are a wonderful gene library for deriving biobricks.And, we can see the enormous protential of other species other than E.coli in energy,health,enviroment…</p><br />
<br/><br />
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<a><img src="https://static.igem.org/mediawiki/2012/0/03/Ouc-human-practiceIMCAS.jpg"/></a><br />
<br/><br />
<br/><br />
<h2>3/iGEM in QIBEBT</h2><br />
<p style="text-decoration:underline;">*What is QIBEBT?</p><br />
<p><span></span>QIBEBT is short for Qingdao Institute of Bioenergy and Bioprocess Technology.Aiming at producing renewable biofuels by plants,QIBEBT utilize synthetic biology approach to engineer a series of energy plants like fuel algae.<br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*Researcher Xu Jian Visited Ouc iGEM Lab</p><br />
<p><span></span> Last year,our instructor,researcher Xu Jian visted our lab and shared his comments on synthetic biology with us,then we reached an agreement of cooperation.<br />
<br/><br />
<br/><br />
<a style="margin-left:70px;"><img src="https://static.igem.org/mediawiki/2012/e/ea/Ouc-human-practice-xujian.jpg"/></a><br />
<br/><br />
<p style="text-align:center;">Our Instructor Researcher Xu Jian Visited Ouc iGEM Lab</p><br />
<br/><br />
<br/><br />
<p style="text-decoration:underline;">*Making iGEM known in QIBEBT</p><br />
<p><span></span>“The question that fresh labmen come up with always concerns with the most basic problem,”says Ma Bo,another instructor of us,”and they always unscarred by experimental depression or old sterotypes,that is the most invaluable idea for our lab.”<br />
<br/><br />
<br/><br />
<a style="margin-left:180px;"><img src="https://static.igem.org/mediawiki/2012/4/4d/Ouc-human-practice-gonginimcas.jpg"/></a><br />
<p style="text-align:center;">Performing Experiments in QIBEBT</p><br />
<br/><br />
<p><span></span>Through we performed most of our experiments in QIBEBT,which focused on synthetic biology engineering,little knowed iGEM including our instructor,they even thought we were playing house like child!We were depressed and determined to change their attitude,to this end, we showed our enthusiasm and sharpness as an iGEMer,many researchers were impressed by the novel idea of iGEM projects,rigorous experimental design and detailed datasheet!<br />
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PengYong
http://2012.igem.org/Team:OUC-China/Modeling/NoiseAnalysis
Team:OUC-China/Modeling/NoiseAnalysis
2012-10-27T01:53:47Z
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<h1 style="border:none;">Noise analysis-model</h1><br />
<p><strong>Aim:</strong><span></span>To figure out whether output is robust when presented with intrinsic noise.<br />
<br/><br />
<br/><br />
<p><strong>Steps:</strong><br />
<br/><span></span>1. Use the Gillespie algorithm to perform the stochastic analysis;<br />
<br/><span></span>2. Draw probability distribution figures (pdf);<br />
<br/><span></span>3. Compute the noise statistically.<br />
<br/><br />
<br/><br />
<p><strong>Background:</strong><span></span>After having determined the parameter range, we would make the noise analysis.According to theoretical predictions, elementary chemical reactions involved in biochemical processes exhibit substantial stochastic fluctuations when low numbers of reactant molecules are involved within the small volume of a living cell. <br />
The existence of significant stochastic fluctuations in biochemical processes has been confirmed by numerous experiments including tracking of individual protein molecules in individual cells in gene expression processes[Zhou L, Gregori G, Blackman J,Robinson J, Wanner B (2005) Stochastic activation of the response regulator PhoB by noncognate histidine kinases.J Integrative Bioinformatics 2: 11.] <br />
We refer to the most classical Gillespie algorithm and lots of improvements have been made by later generation. For example, Sagar Indurkhya et al mentioned that Dynamic Monte Carlo methods are a common means of simulating the time-evolution of chemical systems.The Gillespie Algorithm (SSA) [1] is the standard algorithm for this process, and has inspired a variety of derivative methods that speed up computation, including the Optimized Direct Method (ODM) [2] and the Next Reaction Method (NRM) [3]. These methods, however, are still computationally costly.Now,we still use the classical algorithm.:<br />
<br/><br/><br />
<p>ηij is used to describe the noise, where:<br />
<br/><span></span><br />
<a><img style="margin-left:270px;" src="https://static.igem.org/mediawiki/2012/3/32/Ouc-modeling-math20.JPG" /></a><br />
<br/><br />
<p style="text-align:center;">σ ij stands for standard deviation and <Xi> represents the mean value. If i=j then</p> <br />
<br/><a><img style="margin-left:270px;" src="https://static.igem.org/mediawiki/2012/9/9a/Math21.JPG" /></a><br />
<br/><br />
<p><span></span>The value of noise is ηi2 .<br />
If the mean([m]1:1)-mean([m]1:2) >> the value of noise, we identify the output wouldn’t be drown in noise.<br />
<br/><br />
<br/><span></span><br />
<a><img src="https://static.igem.org/mediawiki/2012/8/89/Ouc-parameter_range.png" /></a><br />
<br />
<h2>Result_Ratio sensor</h2><br />
<br/><br />
<p> <br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/a/a2/Ouc-rs2.png"></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Fig.1 Pdf means probability distribution frequency <br />
</p><br />
<br/><br />
<br/><br />
<p>The statistics is given below:<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/5/52/Ouc-rs_analysis2.png"></a><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<br />
<h2>Comparator</h2><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/b/b3/Ouc-c2.png"></a><br />
<br/><p style="text-align:center; font-size:90%;">Fig.2 Pdf stands for probability distribution frequency.</p><br />
<br/><br />
<br/><br />
<p>The statistics is given below:<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/c/c4/Ouc-c_analysis2.png"></a><br />
<br/><br />
<br/><br />
<br />
<h2>Conclusion</h2><br />
<br/><br />
<p><span></span>Both ratio sensor and comparator, the change of mRNA concentration is robust enough when presented with noise(mean([m]1:1)-mean([m]1:2) >> noise).It is rational to design our device by this parameter sets.<br />
<br/><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Modeling/ParameterSweep
Team:OUC-China/Modeling/ParameterSweep
2012-10-27T01:51:43Z
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<h1 style="border-bottom:none;">Parameter sweep</h1><br />
<br/><br />
<p><strong>Aim:</strong>To discover the optimized ranges of important parameters and get specific directions from simulation heatmap.<br />
<br/><br />
<p><strong>Steps:</strong><br />
<br/><br />
<br/><br />
<a><img style="margin-left:19px;" src="https://static.igem.org/mediawiki/2012/f/fb/Ouc-modeling-math13.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Brief Result:</strong><span></span>We determined the optimal parameter ranges for the ternary system, and selected a best parameter set for noise analysis.<br />
<br/><br />
<br/><br />
<h2>Why choose slope as an indicator?</h2><br />
<p>Slope is given by : Slope= Δ[m]steady/Δratio=2* ([m]steady1:1 - [m]steady1:2)<br />
<br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/6/6e/Ouc-modeling-math14.JPG" /></a><br />
<br/><br />
<br/><br />
<p>Also slope is not the only candidate indicator, the other indicator candidates weeded out are given below:<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/0/0d/Ouc-modeling-math15.jpg" /></a><br />
<br/><br />
<br/><br />
<h2>‘Sensitive’ does not means ‘important’</h2><br />
<br/><br />
<p><span></span>Sensitive does not means important, for sensitive parameters are fixed inherently and are not adjustable, always.<br />
<br/><span></span>We swept 4 parameters for the reasons given below:<br />
<br/><br />
<br/><br />
<a><img style="margin-left:69px;" src="https://static.igem.org/mediawiki/2012/d/d0/Math16.JPG" /></a><br />
<br/><br />
<br/><br />
<h2>Establish parameter sweep database (4 parameters at the same time)</h2><br />
<br/><br />
<p><span></span>We swept km,βm,αm, ks at the same time and collected 120,000 3-ODE steady-state datapoints by mass computation.<br />
<br/><span></span>For the database is a collection of 5-Dimensional data points, it’s rational to show the result in the format of projection on 2-D surface to indicate the relationship between slope and arbitrary 2 parameters we swept<br />
<br/><br />
<br/><br />
<a><img style="margin-left:85px;" src="https://static.igem.org/mediawiki/2012/1/1a/Ouc-modeling-math17.JPG" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Comparator(slope vs. arbitrary 2 parameters)</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:82px;" src="https://static.igem.org/mediawiki/2012/e/eb/Math18.JPG" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Ratio Senor(slope vs. arbitrary 2 parameters)</p><br />
<br/><br />
<h2>Determine optimal parameter range</h2><br />
<br/><br />
<p>We screened the database to get rid of unreasonable datapoints with restrictions:<br />
<br/><br />
<br/><br />
<a><img style="margin-left:90px;" src="https://static.igem.org/mediawiki/2012/7/76/Ouc-modeling-math.JPG" /></a><br />
<br/><br/><br />
<p>We will use the best parameter set to perform noise analysis.<br />
<br/><br />
<br/><br />
<br />
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PengYong
http://2012.igem.org/Team:OUC-China/Modeling/ODEModel
Team:OUC-China/Modeling/ODEModel
2012-10-27T01:49:58Z
<p>PengYong: </p>
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<h1 style="border-bottom:none;">ODE Model</h1><br />
<h2>ODE simulation</h2><br />
<p><strong>Aim:</strong> To verify feasibility of ratio sensor & comparator theoretically<br />
<br/><br />
<br/><br />
<p><strong>Steps:</strong><br />
<p><span></span>1. Establish ODE equations based on Mass-action law;<br />
<br/><span></span>2. Investigate reasonable parameter sets from previous researches;<br />
<br/><span></span>3. Simulation;<br />
<br/><span></span>4. Verify the feasibility of devices.<br />
<br/><br />
<br/><br />
<p><strong>Results:</strong><span></span>Both Ratio Sensor and Comparator are feasible within the affordable range in vivo. However, the parameter space of Ratio sensor is not wide enough, which adds uncertainty on constructing such a device.<br />
<br/><br />
<br/><br />
<h2>Background</h2><br />
<p><span></span>Simulation based on mass-action law is a common method of modeling sRNA regulation[1].<br />
Generally, we perform our simulation under the guidance given below<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/a/a5/Ouc-modelfc.png" /></a><br />
<br/><br />
<br/><br />
<p><span></span>The differences on design between ratio sensor and comparator is noted in the <a href="https://2012.igem.org/Team:OUC-China/Project/DesignMaking/GoalandDesign">project</a> page. <br />
<br/><br />
Considering the RNA pairing reaction as chemical reaction,we will see the probability of collision is determined by their concentration, and the probability of pairing after collision is determined by their affinity(that is reaction constant k in mass-action law).<br />
<br/><br />
<br/><br />
<a><img style="margin-left:25px;" src="https://static.igem.org/mediawiki/2012/8/83/Ouc-project-designmaking-design0.jpg" /></a><br />
<br/><br />
<br/><br />
<p><span></span>Noting that the input(extra inducer concentration) controls alphas & alpham(RNA synthesis rate), we formulated the mechanism above quantitatively via a simple kinetic model(ODE model) for three RNA interaction. The model is cast in terms of two mass-action equations for the cellular concentrations of the sRNA ([s1],[s2]) and its target GFP mRNA ([m]):<br />
<a><img style="margin-left:175px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-designmaking-design3.JPG" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/0/0b/Ouc-project-designmaking-design4.jpg" /></a><br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/6/6a/Ouc-modeling-math3.jpg" /></a><br />
<br/><br />
<br/><br />
<p><strong>Is it feasible?</strong><span></span>In this ternary system,there are at least 6 parameters uncertain,we verified the feasibility of constructing such a device through ODE simulation using parameter set from previous researches, fortunately,it is viable theoretically within the affordable parameter range in cell, and the apparent enough with the presence of noise<br />
<br/><br />
<br/><br />
<a><img style="margin-left:80px;" src="https://static.igem.org/mediawiki/2012/5/59/Ouc-project-designmaking-design6.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Comparator</strong><span></span>For comparator, simple conclusions could be drawn:<br />
<br/><br />
<br/><br />
<p style="text-align:center;"> When as1/as2>1, [m]steady state=0;<br />
<br/> When as1/as2<1, [m]steady-state=60</p><br />
<br/><br />
<br />
<p><span></span>Considering the mechanism of comparator as a molecular titration,we also expect a ultra-sensitivity behavior at the switch point(as1/as2=1),which will contribute to the “comparing” decision-making behavior.<br />
<br/><br />
<br/><br />
<p><strong>Ratio sensor</strong><span></span>For ratio sensor, an apparent peak can be observed when as1/as2=1<br />
We run a supplementary simulation at point of as1:as2=1, making comparisons when as1:as2=1:1 and as1:as2=1:2(Fig.3)<br />
<br/><br />
<br/><br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/1/1d/Ouc-mo1.jpg" /></a><br />
<br/><br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/c/c7/Ouc-mo2.jpg" /></a><br />
<br/><br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/f/f8/Ouc-mo3.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="font-size:90%; text-align:center;">Fig 3.</p><br />
<br/><br />
<br/><br />
<p><span></span>Discussion: The three differential equations are established based on Erel Levine et al and we have made a further development. In addition, our model has changed from the one for single interaction between sRNA and mRNA to the one for three kinds of interaction (s1RNA and s2RNA, s1RNA and mRNA, s2RNA and mRNA ). Therefore the latter model would come as a meaningful example for researchers to explore more interactions among RNAs. Of course, we can get the model, to a large degree, due to the creative achievement of Erel Levine et al and others.<br />
<br/><br/><br />
<p><strong>References</strong><br />
<br/>[1] Levine, E., et al., Quantitative characteristics of gene regulation by small RNA. PLoS biology, 2007. 5(9)<br />
<br/>[2] Nicolas E. Buchler1, Matthieu Louis, Molecular Titration and Ultrasensitivity in<br />
Regulatory Networks, J. Mol. Biol. (2008) 384, 1106–1119 <br />
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<h1 style="border-bottom:none;">Discussion</h1><br />
<br />
<p><span></span>The results showed that some of our speculations include that the <i>gvp</i> genes in prokaryotes of generality. The Cyanobacteria <i>gvp</i> gene exists corresponding identification σ factor in escherichia coli in causing <i>gvp</i> gene can be in escherichia coli to the normal expression. ( <a href=” http://www.vcu.edu/cyanonews/V15/promoter.html”>Wipa Chungjatupornchai</a>) on the other hand when we transformed the recombined gene cluster and the two structure parts <a href="http://partsregistry.org/Part:BBa_K737006">BBa-K737006</a> and <a href="http://partsregistry.org/Part:BBa_K737007">BBa-K737007</a> into the Expression strains Top10 it can also make floating bacteria in the layer suface.( Figure22). The results show that although <i>gvp</i> gene cluster has some unknown control sequence, however these control sequence will not effect on gas vesicle structure protein assembly. When we prove the gvp gene is relying on electrostatic interactions, salt bridges and van der Waals forces to maintain assembly.[ 7] Through our experiment once again proven that the gas vesicle structure between the two structural protein assembly don't need some control sequence exist in the genome.in conclusion the regulation that greatly enhanced the gas vesicle gene scalability in applied field. in most cases majority of foreign protein expression in escherichia coli expression system may cause metabolic burden to form some insoluble aggregate termed “inclusion”.[8] Through our experiment also proven that the gas vesicle protein could not in <i>E.coli</i> expression system. </p><br />
<br/><br/><br />
<h1 style="border:none;">Future Work</h1><br />
<h2 style=>Gas Vesicle</h2><br />
<br/><br />
<p><span></span>Though we have done a lot of work, there still are some works to do in the future. We want to regulate the ratio of GvpA and GvpC to make <i>E.coli</i> floating better. In one hand, we ligate two inducible gene circuits with <i>gvp</i> genes, regulate the expression of Gvp protein and detect the expression of Gvp by detecting the fluorescence of GFP. On the other hand, we make fusion protein GvpC:GFP to detect the expression of GvpC.<br />
<br/><br/><span></span>Next, we want to get the 2kb length of <i>gvp</i> gene cluster which contain the <i>gvp</i>C-16 gene in addition. We think it may make <i>E.coli</i> buoyant better. As for experiment of Gas vesicle protein quantitative analysis, we will establish GFP protein expression quantity and the relationship between the Fluorescence Unit. Now, we have ligated the inducible gene circuits. Later, we will examine them by inducing with IPTG or aTc.<br />
<br/><br/><span></span>In addition to the above discussion and the project to do, we also believe that project in gas vesicle aspect has lots to expand space for example through the cell floating in the sea surface and produce algal blue element to form a similar filter to restrain the effect of red tide algae growth, or cell floating in the sea surface release inhibition of red tide algae growth of toxins to restrain the growth of red tide algae. Even we can make cells in Marine thermocline and halocline gathering in some nutrient elements through the cell floatation will nutrient element to the sea surface.<br />
<br/><br />
<br/><br />
<h2>Phosphate Accumulation</h2><br />
<p><strong>Background</strong></p><br />
<br/><p><span></span>Inorganic phosphate (Pi) is recognized as one of the major nutrients contributing to the outbreak of red tide. So we want our RNA comparator not only to be a receptor but also an actuator that can accumulate excess Pi in the form of polyphosphate (polyP) from water when the <i>E.coli</i> senses the concentration of phosphate over the warning limit. An enzyme responsible for polyP synthesis is the homotetrameric polyP kinase (PPK). PPK polymerizes the terminal phosphate of ATP into poly. [9]Acetate kinase (<i>ack</i>A) was employed as an ATP regeneration system for polyphosphate synthesis. Researchers have demonstrated Strain MV1184 containing pEP03 (<i>ppk</i> and <i>ack</i>A ) showed the highest rates of Pi removal from the medium.</p> <br />
<br/><br />
<a><img style="margin-left:20px;" src="https://static.igem.org/mediawiki/2012/2/2c/Ouc-de0.png" /></a><br />
<br/><br />
<p style="margin-left:350px; margin-top:-500px; width:350px;">This recombinant strain removed approximately 90% of the Pi from the medium within 4 h. The data of this result is very satisfactory. Although another recombination strain contain pEP022+pBC29 (<i>ppk</i> and <i>pst</i> operon) has better effect after six hours [10], the pst operon is too long to be used, so we choose to create the recombination strain containing ppk and ackA that we standardize by ourselves.</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:340px; margin-top:-10px;" src="https://static.igem.org/mediawiki/2012/6/61/Ouc-phosphate1.jpg" /></a><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<p><strong>Design and Experiment</p></strong><br />
<p><span></span>To get standardized parts, an entire <i>ack</i>A gene fragment was amplified by PCR with the primers designed by ourselves from the mutation of <i>E.coli</i> K12 strain, DH5αand inserted into the PSB1C3 vector. <i>ppk</i> contains an EcoRI enzyme site need to be mutanted. So we got it from the <i>E. coli</i> DH5αchromosome and inserted it into pGEM-Tvector, then cut it in XbaI and PstI and inserted the fragment into PSB1C3 cutting by XbalI and PstI sites first.<br />
We ligated the two parts with strong promoter and terminator and transferred them into high-copy-number vector to test the ability of recombination strain to accumulate inorganic phosphate (Pi).We would like to monitor the change of Pi in the medium using an autoanalyser AA3(Bran+Luebbe)applying the standard colorimetric procedures(Koroleff, 1983). <br />
</p><br />
<br/><br />
<a><img style="margin-left:245px;" src="https://static.igem.org/mediawiki/2012/b/b5/Ouc-de1.png" /></a><br />
<br/><br />
<br/><br />
<p><strong>Future Work</p></strong><br />
<p><span></span>We also want to control the gene dosage of the two parts by ligating them with Lac promoter and Tet promoter, so we can monitor whether the ability of <i>E.coli</i> to absorb the Pi in the medium would have change and how would it change by adding different amount of IPTG and arabinose. <br/><br />
<br/><br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/4/4f/Ouc-de2.png" /></a><br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/5/5d/Ouc-de3.png" /></a><br />
<br/><br />
<br/><br />
<h1 style="border-bottom:none;">References</h1><br />
<p>1. A.E. Walsby (March 1994). "Gas Vesicles". Microbiological reviews 58(1): 94-144<br />
<br/>2. Steven J. Beard, Barbara A. Handley, Anthony E. Walsby Spontaneous mutations in gas vesicle genes of Planktothrix spp. Affect gas vesicle production and critical pressure FEMS Microbiology Letters 215 (2002) 189~195<br />
<br/>3. P. K. Hayes, B. Buchholz, and A. E. Walsby Arch Microbiol Gas vesicles are strengthened by the outer-surface protein <i>gvp</i>C (1992) 157:229 234 <br />
<br/>4. P. K. Hayes. R. S. Powell Arch Microbiol The <i>gvp</i>A/Ccluster of Anabaena flos-aquae has multiple copies of a gene encoding <i>gvp</i>A (1995) 164 :50-57<br />
<br/>5. Jeff Hasty , David McMillen, & J. J. Collins Engineered gene circuits NATURE VOL 420 14 NOVEMBER 2002<br />
<br/>6. oliver scholz, Anja Thiel, wolfgang Hillen and Michael Niederweis Eur.J.Biochem, Quantitative analysis of gene expression with an improved geen fluorescent protein 267,1565-1570 (2000) FEBS 2000<br />
<br/>7. Hussein M. Ezzeldin a, Jeffery B. Klauda b, Santiago D. Solares Modeling of the major gas vesicle protein, <i>gvp</i>A: From protein sequence to vesicle wall structure Journal of Structural Biology 179 (2012) 18–28<br />
<br/>8. Vasina JA, et al, Expression of aggregation prone recombinant, parteins at lour temperatures a comparative study of the <i>Escherichia coli</i> <i>csp</i>A and tac promoter systems protein Expr purify 1997.9: 211-218<br />
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9. Gregory D. Davis,* Claude Elisee, Denton M. Newham, Roger G. Harrison New Fusion Protein Systems Designed to Give Soluble Expression in Escherichia coli BIOTECHNOLOGY AND BIOENGINEERING, VOL. 65, NO. 4, NOVEMBER 20, 1999<br />
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10. Ahn, K., and A. Kornberg. 1990. Polyphosphate kinase from <i>Escherichia coli</i>. J. Biol. Chem. 265:11734-11739.<br />
<br/>11. Unichi Kato, Katsufumi Yamada, Ayako Muramatsu, Hardoyo, Hisao Ohtake.1993. Genetic Improvement of Escherichia coli for Enhanced Biological Removal of Phosphate from Wastewater. Microbiology, 59(11):3744-3749<br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/GVP/DesignandResult
Team:OUC-China/Project/GVP/DesignandResult
2012-10-27T01:43:56Z
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<h1 style="border-bottom:none;">Design and Result</h1><br />
<h2>Cloning strategy</h2><br />
<p><strong>1. Gene cluster</strong><span></span>For our <i>gvp</i> gene cluster we use the specificity primer form Walsby 2002 and add the necessary restriction sites that annealed these pieces of DNA have EcoRI and SpeI sticky ends. We use the vector psb4K5 because the uncoordinated expression of the entire <i>gvp</i>A/C cluster from its own constitutive promoter when carried on a high-copy number vector could result in the accumulation of lethal quantities of gas vesicle proteins in the host cells.Hence we use the low-copy number vector to ensure that the gene cluster can in the host to the normal expression.[3] so we use The vector pSB4K5 containing <i>gvp</i> gene cluster was cut with EcoRI and PstI and was ligated with B0015. ( Figure 4 5) After PCR inspection by using Specific primer we get the target fragment. ( Figure7)<br />
On the other hand, In order to prove that the integrity of the <i>gvp</i> gene cluster we use a set of genetic circuit controlled by the methods OMPR promoter reverse connection in the back of the K73710 using another light controlled element in laser under excitation of start OMPR promoter that the expression of gene cluster is restrained to prove <i>gvp</i> gene cluster integrity. ( Figure 23)<br />
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<a><img src="https://static.igem.org/mediawiki/2012/c/c2/Ouc-project-gvp3.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 4 This 1 kbp part contains 2 open reading frames coding for gas vesicle genes (<i>gvp</i>A and <i>gvp</i>-20) and the <i>Cyanobacteria</i> gene promoter and RBS (BBa_K737019 and BBa_K737020) from <i>Bacillus Planktothrix rubescens</i> strain BC-Pla 9402. Promotion of the sequence results in expression of gas vesicles, organelles made entirely out of protein. These organelles contain gas and therefore provide buoyancy to the cell. <br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:112px;" src="https://static.igem.org/mediawiki/2012/7/7c/Ouc-project-gvp4.jpg" /></a><br />
<a><img style="margin-left:93px;" src="https://static.igem.org/mediawiki/2012/5/55/Ouc-project-gvp5.jpg" /></a><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 5 and 6The cloning strategy of buoyancy device and light control device. Because the uncoordinated expression of the entire <i>gvp</i>A/C cluster with its own constitutive promoter could result in the accumulation of lethal quantities of gas vesicle proteins when carried on a high-copy number vector in the host cells. <br />
</p><br/><br />
<a><img style="margin-left:50px;" src="https://static.igem.org/mediawiki/2012/a/af/Ouc-gvp2.jpg" /></a><br />
<p style="margin-left:360px; margin-top:-280px;">Use the low-copy number vector pSB4A to ensure that the gene cluster can in the normal expression in the host<br />
The light control gene circuit from our iGEM lab is based on UC-iGEM 2005.we use part BBa_ I15008 (ho1) and BBa_I15009 and Cph1 / EnvZ FP redesigned photosensitive gene line, fixed iGEM part library of the BBa_I150010 Cph1 / EnvZ FP mistakes, successful synthesis of 660 nm red light sensitive gene expression device, which in the red light input conditions closed gas vesicle expression. (Figure7)<br />
<br/></p><br />
<br/><br />
<p style="font-size:90%; margin-top:50px;">(Figure7) the 660 nm laser induced device<br />
It is made up of three 660 nm wavelength of<br />
laser device and a cooling fan composition can<br />
be fixed a root tube that nutrient solution to<br />
get full sunshine has reached laser induced effect<br />
( OUC-China team)</p><br />
<br/><br />
<br/><br />
<br/><br />
<p><strong>2.<i>gvp</i>A and <i>gvp</i>C genes</strong><span></span>It’s out second step to take out the <i>gvp</i>A and <i>gvp</i>C gene from <i>Planktothrix rubescens</i> 9402 strain (CCAP 1460/9). Since there is no sequence information in NCBI about Pla 9402, we decided to take advantages of its translocation mechanism. So we use the Pla 9401 (GenBank: AJ132354.1) and Pla 9402/b (GenBank: AJ494991.1) gene sequence as the template to design primers for <i>gvp</i>A, <i>gvp</i>C-20psi and <i>gvp</i>C-16omega. In consideration of Pla 9402’s repetitive sequences of gvp gene, we took two different approaches to take out the genes: one is directly doing PCR with the genome of Pla 9402 as template, while the other is PCRed on the BssSI-enzymed template of Pla 9402 genome. The gene <i>gvp</i>A and <i>gvp</i>C-20psi was successfully cloned through both these pathways, but the attempts to clone <i>gvp</i>C-16omega gene failed. <br />
<br/><br />
<br/><br />
<br/><br />
<p><strong>3.Synthetic <i>gvpA/C</i> gene cluster</strong><br />
<span></span>In order to realize the gas vesicle as the decision making output actuator, we clone the gvp structure gene gvpA and gvpC from the natural gvp gene cluster and ligate the promoter(<a href="http://partsregistry.org/Part:BBa_J23106">BBa-J23106</a>)RBS(<a href="http://partsregistry.org/Part:BBa_B0034">BBa-B0034</a>)and terminator(<a href="http://partsregistry.org/Part:BBa_B0015">BBa-B0015</a>)of the E.coli expression system. Because we use the standardized promoter and RBS in this part, so this part can be used in more expression system and control its expression by ourselves as the decision making output actuator. It may extend the applicability of gvp floating device.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:97px;" src="https://static.igem.org/mediawiki/2012/7/77/Ouc-gvp4.png" /></a><br />
<p style="font-size:90%; text-align:center;">Fig 7: We want to recombine the gvpA/C cluster and control its expression by ourselves to realize the decision making output.</p><br />
<br/><br />
<a><img style="margin-left:160px;" src="https://static.igem.org/mediawiki/2012/b/bc/Ouc-gvp4.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="font-size:90%; text-align:center;">Fig 8:the expression situation of th e Gas vesicle synthetic gene cluster in escherichia coli JM109,from the picture, we can see obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon.</p><br />
<br/><br />
<br/><br />
<h2>Floating <i>E.Coli</i></h2><br />
<br/><br />
<p><span></span> We transformed 1Kbp <i>gvp</i> gene cluster into JM109 and stationary culture for 36 hours. You can find the E.Coli floating on the surface! (Figure 10 and 11)<br />
<br/><span></span>The expression situation of the Gas Vesicle gene cluster in Escherichia coli JM109, from the picture, we can see obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon. The picture on the right of the experimental group were liquid surface layer and intermediate layer of fluid sampling results, we can see that the fluid layer surface bacteria volume density is obviously higher than that of the middle layer of fluid.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:110px;" src="https://static.igem.org/mediawiki/2012/9/97/Ouc-project-gvp7.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 10 and 11. The expression situation of the Gas vesicle gene cluster in <i>Escherichia coli</i> JM109, from the picture, we can see obviously gas vesicle due to the expression of bacteria produce clear separation phenomenon. The picture on the right of the experimental group were liquid surface layer and intermediate layer of fluid sampling results, we can see that the fluid layer surface bacteria volume density is obviously higher than that of the middle layer of fluid.<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span> The buoyancy of Gvp was tested by using the buoyancy test protocol. The cells were grown in a test tube with LB medium for 36 hours (Figure 10) and were left for a while before detected in order to give the cells time to sink or float.<br />
Trace stratified take liquid apparatus. It is made up of a 10 ml volumetric syringe and five a 1 ml volumetric syringe composition. It can be used as a culture cell containers and can be performed every layer of the synchronous sampling the aim is to avoid the sampling process caused by vibration caused by fluid layer surface thallus under. The whole device can make quantitative analysis more accurate.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:170px;" src="https://static.igem.org/mediawiki/2012/c/cb/Ouc-project-gvp8.jpg" /></a><br/><br/><br />
<p style="text-align:center; font-size:90%;">Figure 12. Trace stratified take liquid apparatus. It is made up of a 10 ml volumetric syringe and five a 1 ml volumetric syringe composition. It can be used as a culture cell containers and can be performed every layer of the synchronous sampling the aim is to avoid the sampling process caused by vibration caused by fluid layer surface thallus under. The whole device can make quantitative analysis more accurate. (OUC-China team)<br />
</p><br />
<br/><br />
<br/><br />
<br/><br />
<p><span></span> Because when the bacteria die, it may float on the surface. There is a question, do the bacteria floating on the surface die or not So we made a microscopic examination. We use a vital stain to examine the bacteria die or not. Dead bacteria can’t decompose the stain, so they appear blue, while the living cells turn to be transparent (Figure 11 and 12).<br />
<br/><br />
<br/> <br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/9/9e/Ouc-project-gvp9.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 13 and 14. The left picture shows that most of surface bacteria are living because there are few blue spot here. The right picture is dead bacteria as control. We can see that, almost all the bacteria are dead.<br />
</p><br/><br />
<br/><br />
<br />
<a><img style="margin-left:72px;" src="https://static.igem.org/mediawiki/2012/6/6f/Ouc-gvp10.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 15 and 16. Under the microscope, we use the blood count method to observe the different liquid layer cell capacity, the left is from the liquid layer on the surface of the samples taken from the experimental group, and the right is middle layer of fluid samples. Through the under the microscope observation can obviously observed as the cells expressed in the gas vesicle that cells in the culture solution of concentrated in the surface layer of fluid.<br />
</p><br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/d/d2/Ouc-project-gvp11.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 17 and 18. Under the microscope we use the blood count method to observe the different liquid layer cell capacity, the cell from the bottom of the control samples taken from control group(left)the surface layer of fluid samples.(right) Because the control group cells without expression of Gas vesicle that the force of gravity has a large number of somatic settlement in the test tube through the bottom under the microscope observation we can clear observation the samples of bottom and the difference between the surface sample.</p><br />
<br/><br />
<br/><br />
<p><span></span> According to the different layer accurately sampling, the samples blood count determine different height body effusion layer cell number so as to reflect the situation of gas vesicle expression. The results were best visible with and cell density of OD600=2.382 in Liquid surface. And we find that in the test tube, in addition to liquid layer surface and bottom cell is dense in the rest of the liquid layer densities and roughly the same and surface bacteria volume density difference of an order of magnitude. From the control group of counting the results to the distribution of cells with normal cells in the test tube, the distribution so can also explain the experimental results is due to gas vesicle expression in cells. We also do each liquid layer absorbance inspection results and cell number experimental results high similarity from the side proved the correctness of blood count experiment.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:79px;" src="https://static.igem.org/mediawiki/2012/3/3e/Ouc-ode1.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 19. The picture shows the gas vesicle in the cells after expressed in the overall trend. We in the cultivation of the cells into logarithmic phase after transferring them to test tube, quiet place and culture 36 hours after using Trace stratified take liquid apparatus at the same time to each liquid layer for sampling. Then each layer of fluid samples of blood count the number of cells with statistics. Results show that gas vesicle due to the existence of the cell concentration in the test tube surface layer of fluid, and control group due to the action of gravity that most cells sedimentation in the test tube bottom.<br />
</p><br/><br />
<br/><br />
<a><img style="margin-left:79px;" src="https://static.igem.org/mediawiki/2012/2/25/Ouc-ode2.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="text-align:center; font-size:90%;">Figure 20. Graph of OD measurements at 600nm of both experience group and control group. From the experimental results can be seen in the liquid surface can see cell number have significant abrupt transformation.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:10px;" src="https://static.igem.org/mediawiki/2012/7/7e/Ouc-project-gvp14.JPG" /></a><br />
<a><img style="margin-left:370px; margin-top:-520px;" src="https://static.igem.org/mediawiki/2012/7/74/Ouc-project-gvp15.JPG" /></a><br />
<p style="margin-left:400px; margin-top:-100px; font-size:90%">Figure21. Fitting function that function relation is y=25300x4-4726000x3+30515000x2-7.7444000x+70902000. In the picture X axis representative distance test tube bottom every centimeter liquid column and ordinate for cell number.</p><br />
</p><br/><br />
<br/><br />
<br/><br />
<br/><br />
<p><span></span>Through experiment data f and significant t test. We draw conclusion that in the experimental group and control group on the surface of the liquid layer cell count there exist significant differences this difference is due to the experimental group cells express gas vesicle.<br />
<br/><br />
<br/><a><img style="margin-left:10px; margin-top:-3px;" src="https://static.igem.org/mediawiki/2012/d/d6/Ouc-project-gvp16.JPG" /></a><br />
<p style="margin-left:140px; margin-top:-310px; width:600px;"><span></span>Besides detecting OD/600, we use blood counting method to get cell number in different water-courses. With the help of MATLAB analysis,we can get fitting function of cell number in different water-courses.<br />
<br/><br />
<br/><span></span>Fitting function that function relation is y=25300x4-4726000x3+30515000x2-7.7444000x+70902000. In the picture X axis representative distance test tube bottom every centimeter liquid column and ordinate for cell number.<br />
<br/><br/><span></span>After getting the plasmids of constitutive GvpA and GFP generator, constitutive GvpC and GFP generator, we transform the two plasmids into E.Coli to see if they can float. As expected, they float! It shows that the <i>gvp</i>A and <i>gvp</i>C genes work. In other word, only two genes (about 700bp) can make bacteria floating.<br />
<br/><br/><br />
<p style="font-size:90%; margin-left:10px;"> Figure 22.Floating bacteria with double plasmids.</p><br />
<br/><br />
<br/><br />
<h2>Gas vesicle protein and Expressive inspection</h2><br />
<br/><br />
<p><span></span>We cloned the <i>gvp</i> gene cluster and <i>gvp</i> coding gene form <i>Planktothrix rubescens</i>, which can make the protein GvpA and GvpC-20.The two types of gas vesicle structure protein can be assembled into an empty bubble structure of the Protein polymers which can be in the electron microscope observation under.<br />
<br/><span></span>After successfully transform the BBa_K737010 into the Expression strains JM109, the experiment results show that gas vesicle gene can be expressed in <i>E.coli</i> when gas vesicle gene expression in cells it can make floating bacteria in nutrient solution layer surface. The results showed that some of our speculations include that the <i>gvp</i> genes in prokaryotes of generality.<br />
<br/><br />
<br/><br />
<a><img style="margin-left:4px;" src="https://static.igem.org/mediawiki/2012/d/d2/Ouc-project-gvp17.JPG" /></a><br />
<br/><br/><br />
<p style="font-size:90%; width:200px; margin-left:20px; margin-top:-10px;">Figure 23. Gas vesicles in a freeze-fractured cell of Prosthecomicrobium<br />
Pneumaticum showing the straight –sided cone (Micrograph by<br />
D. Branton and A.E. Walsby) Magnification 210000x)<br />
<p style="font-size:90%; width:200px; margin-left:250px; margin-top:-160px;">Figure 24.Gas vesicles in E.coli protoplasts (BBa_K190033).<br />
The cells were treated with Lysozyme and SDS to<br />
create the protoplasts, uranyl acetate was used for<br />
staining. Magnification: 11500x.(2009 Groningen iGEM )<br />
<p style="font-size:90%; margin-top:-150px; width:250px; margin-left:470px;">Figure 25.The <i>gvp</i> gene cluster in 1% agarose gel form 1 to 4<br />
1 and 2 representative gvp gene cluster + B0015 + R0082 + B0034<br />
3 and 4 gvp representing gene cluster + B0015 and gvp gene cluster<br />
</p><br />
<br/><br />
<br/><br />
<br/><br />
<br/><br />
<p><span></span>To check whether gas vesicles really were present in the cells we did some electron microscopy. In Figure 28 a picture of gas vesicles in a protoplast can be seen. This protoplast comes from an <i>E. coli</i> cell that contained a plasmid with the <i>gvp</i> gene cluster (<a href="http://partsregistry.org/Part:BBa_K737010">BBa_K737010</a>). Figure 29 a picture of blank plasmid in the <i>E. coli</i> JM109 in electron microscope.<br />
</p><br />
<br/><br />
<a><img style="margin-left:220px;" src="https://static.igem.org/mediawiki/2012/4/4f/Ouc-gvp1.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="font-size:90%; text-align:center;">Figure 26. Gas vesicles in <i>E. coli</i> protoplasts (<a href="http://partsregistry.org/Part:BBa_K737010">BBa_K737010</a>). The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: 60k x.</p><br />
<br/><br />
<a><img style="margin-left:220px;" src="https://static.igem.org/mediawiki/2012/b/bb/Gvp2.jpg" /></a><br />
<br/><br />
<br/><br />
<p style="font-size:90%; text-align:center;">Figure 27. A blank plasmid in the <i>E. coli</i> JM109 The cells were treated with Lysozyme and SDS to create the protoplasts, uranyl acetate was used for staining. Magnification: x 60k</p><br />
<br/><br />
<br/><br />
<h2>Gas vesicle protein quantitative analysis</h2><br />
<br/><br />
<p><span></span>In order to quantitative analyze the expression of gvp gene inside the cell, we design a genetic circuit that could be realized by inserting the <i>gvp</i> gene alongside GFP as a polycistron[5]. Using this part, we can know the expression level of Gvp protein by the detecting the fluorescence of GFP.It will be transcribed into an mRNA and the two genes each have a same promoter (BBa_ J23106). So we can through the expression of GFP to detect gvp gene expression of roughly quantitative. GFP expression can through the determination expression of the specific excitation wavelength of fluorescence value to calculate. [5][6]<br />
<br/><span></span>We made 2 polycistron circuits of gvpA/C and gfp genes. <br />
<br/><span></span>Our devices are:<br />
<br/><span></span>Constitutive GvpA and GFP generator (BBa_K737006):<br />
<br/><span></span>Promoter (BBa_J23106) + RBS (BBa_B0034) + gvpA + RBS + GFP + terminator (BBa_E0840)<br />
<br/><span></span>Constitutive GvpC and GFP generator (BBa-_K737007):<br />
<br/><span></span>Promoter (BBa_J23106) + RBS (BBa_B0034) + gvpC + RBS + GFP + terminator (BBa_E0840)<br />
<br/><br />
<br/><br />
<a><img style="margin-left:140px;" src="https://static.igem.org/mediawiki/2012/2/25/Ouc-project-gvp18.JPG" /></a><br />
<br/><br />
<br/><br />
<p><span></span>In order to check out the expression of polycistronic genetic circuit .We use the SDS-PAGE(protocol)to get the intracellular total protein. Here is the result of SDS-polyacrylamide gel electrophoresis. (Figure22) We can see GFP and GvpC protein directly. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper blue and red boxes are in the position about 26KD are GFP protein (25.8KD). The lower red box in the position of about 20KD is GvpC-20 (20KD). Because the MW(molecular weight) of GvpA protein is too small, it’s very difficult to see it on the SDS-polyacrylamide gel. But the expression of GFP suggests it may be expressed.<br />
<br/><span></span>Some papers say that GvpA is difficult to see is that GvpA is too hydrophobic to run out of sample hole. Because the gas vesicle is multimodal protein so it cannot isolate by using SDS-PAGE, too. (Figure28)<br />
<br/><span></span>Finally, we consider that GVP as exogenous protein may be in escherichia coli form inclusion body so we through the Wilkinson–Harrison solubility model to predict GVP protein solubility and judgment GVP protein formation the possibility of inclusion body.<br />
<br/><span></span>The revised Wilkinson–Harrison solubility model involves calculating[9]<br />
<br/><span></span>a canonical variable (CV) or composite parameter for the protein for which<br />
<br/><span></span>the solubility is being predicted. The canonical variable in the two parameter model is defined as:<br />
<br/><br/><br />
<a><img style="margin-left:190px;" src="https://static.igem.org/mediawiki/2012/1/15/Ouc-project-gvp19.JPG" /></a><br />
<br/><br/><br />
<p>where: n 4 number of amino acids in protein<br />
<br/>N,G,P,S 4 number of Asn, Gly, Pro, or Ser residues, respectively.<br />
<br/>R,K,D,E 4 number of Arg, Lys, Asp, or Glu residues, respectively.<br />
<br/>λ1 andλ2 coefficients (15.43 and 29.56, respectively)<br />
<br/>The probability of the protein solubility is based on the parameter CV-CV8,<br />
<br/>where CV8 is the discriminant equal to 1.71. If CV-CV8 is positive, the<br />
<br/>protein is predicted to be insoluble, while if CV-CV8 is negative, the<br />
<br/>protein is predicted to be soluble. The probability of solubility or insolubility can be predicted from the following equation:<br />
<br/>Probability of solubility or insolubility =<br />
<br/><br />
<a><img style="margin-left:220px;" src="https://static.igem.org/mediawiki/2012/4/4f/Ouc-project-gvp20.JPG" /></a><br />
<br/><br />
<p><span></span>The Calculation results show that the gas vesicle protein is insoluble protein.it may be form the <br />
inclusion body in the escherichia coli expression system.( Table 2)<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/9/90/Ouc-project-gvp21.JPG" /></a><br />
<br/><br />
<br/><br />
<a><img style="margin-left:280px" src="https://static.igem.org/mediawiki/2012/1/17/Ouc-project-gvp22.jpg" /></a><br />
<p style="margin-left:20px; margin-top:20px; width:700px; font-size:90%;">Figure 28.the result of 10% SDS-polyacrylamide gel electrophoresis.<br />
the expression of protein gas vesicle in E. coli JM109 analyzed by<br />
SDS-PAGE and quantitative measurement. Equal amount of sample<br />
from cell lysate was fractioned into the pellet and subjected<br />
to SDS-PAGE. 1 and 2 bands in pellet represent respectively the<br />
JM109 blank control and import GVP gene cluster strains.<br />
</p><br />
<br/><br/><br />
<a><img style="margin-left:100px;" src="https://static.igem.org/mediawiki/2012/b/b6/Ouc-project-gvp23.JPG" /></a><br />
<br/><br/><br />
<p style="text-align:center; font-size:90%;">Figure 29.The result of SDS-polyacrylamide gel electrophoresis. Lane 4 is top10 control group, lane 3 is constitutive GvpA and GFP generator, lane 5 is constitutive GvpC and GFP generator. The upper blue and red boxes are in the position about 26kDa are GFP protein (25.8kDa). The lower red box in the position of about 20kDa is GvpC-20 (20kDa).<br />
</p><br />
<br/><br />
<br/><br />
<p><span></span>Because the GvpA protein can’t be seen on the SDS-polyacrylamide gel electrophoresis, we wanted to use GFP to detect the expression of GvpA. We use a polycistron of gvpA (gvpC) and gfp to detect the RFU. We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control. (Figure26)First, the RFU of these parts are rather high. Second, the E.Coli with these two plasmids can float. Third, we can see GvpC on the SDS-polyacrylamide gel. So, we can see the two parts can express Gvp Protein and work well.<br />
<br/><br />
<br/><br />
<a><img style="margin-left: 120px;" src="https://static.igem.org/mediawiki/2012/7/7d/Ouc-project-gvp24.JPG" /></a><br />
<br/><br />
<p style="margin-left:20px; margin-top:10px;margin-bottom:20px; font-size:90%;">Figure 30.We can see that, the RFU of Gvp and GFP Generator is about 2000, and the RFU of Gvp and GFP Generator is about 4000, while top 10 is about 0 as control. <br />
</p><br />
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PengYong
http://2012.igem.org/Team:OUC-China/Project/DesignMaking/Result
Team:OUC-China/Project/DesignMaking/Result
2012-10-27T01:41:05Z
<p>PengYong: </p>
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<div class="list-text"><br />
<h1 style="border-bottom:none;">Result</h1><br />
<h2>Device testing <a href="https://2012.igem.org/Team:OUC-China/protocol"><i>Protocol</i></a><br />
</h2><br />
<p><strong>galK::GFP Generator under induction,<br />
surface fitting using MATLAB sftool:<br />
</strong><br />
<p><span></span>galK::GFP_Generator without expressing <i>spot42</i> could be repressed by IPTG/aTc in an unclear way. One assumption is that the cytotoxicity of inducers impairs the capacity of expressing protein.<br/><br />
<br/><br />
<a><img style="margin-left:32px;" src="https://static.igem.org/mediawiki/2012/5/51/Ouc-project-decisionmaking-result0.JPG" /></a><br />
<br/><br />
<br/><br />
<br />
<p><strong>Comparator characterization</strong><br />
<br/><br />
<p><span></span><i>srlA</i> surface fitting using MATLAB sftool:<br />
<br/><br />
<br/><br />
<a><img src="https://static.igem.org/mediawiki/2012/5/5b/Ouc-com.png" /></a><br />
<br/><br />
<br/><br />
<a><img style="margin-left:32px;" src="https://static.igem.org/mediawiki/2012/4/4f/Ouc-project-desicionmaking-result1.JPG" /></a><br />
<br/><br />
<br/><br />
<p><i>ytfJ</i> surface fitting using MATLAB sftool:<br />
<br/><br />
<br/><br />
<a><img style="margin-left:32px;" src="https://static.igem.org/mediawiki/2012/6/63/Ouc-project-decisionmaking-result2.JPG" /></a><br />
<br/><br />
<br/><br />
<p><i>nanC</i> surface fitting using MATLAB sftool:<br />
<br/><br />
<br/><br />
<a><img style="margin-left:32px;" src="https://static.igem.org/mediawiki/2012/f/fc/Ouc-project-desicionmaking-result3.JPG" /></a><br />
<a><img style="margin-left:32px;" src="https://static.igem.org/mediawiki/2012/4/45/Ouc-project-desicionmaking-esult4.JPG" /></a><br />
<a><img style="margin-left:32px;" src="https://static.igem.org/mediawiki/2012/2/2f/Ouc-project-decisonmaking-result5.JPG" /></a><br />
<br/><br />
<br/><br />
<p><strong>Ratio sensor characterization</strong><br />
</br><br />
Secondary architecture of spot42 with artificial region:<br />
</br><br />
<a href="https://2012.igem.org/Team:OUC-China/Modeling/ODEModel"><br />
<img src="https://static.igem.org/mediawiki/2012/6/60/Ouc-rpos-spot42.png" /></a><br />
<br />
</br><br />
</br><br />
Engineered Spot42 keeps repression efficiency:<br />
</br><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/6/66/Ouc-decision1.jpg" /><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/a/a4/Ouc-decision2.jpg" ></a><br/><br/><br />
<p style="font-size:90%; text-align:center;">Fig.components of ratio sensor is tested to show the fused sRNA keeps repression efficiency.the first figure shows that version of RpoS::spot42 keeps nearly half of repression rate. The second one is the version of orS2::spot42(mutant), which shows relatively weaker repression efficiency. The reason might be that 5 bases mutant of spot42 give a weaker pairing of spot42(mutant) and galK. But it is negligible or even better for our design.<br />
<br />
<p><strong>Platform data</strong><br />
<br/><br />
<br/><br />
<a><img style="margin-left:85px;" src="https://static.igem.org/mediawiki/2012/8/85/Ouc-project-discussion0.jpg" /></a><br />
<br/><p style="text-align:center; font-size:90%;">Fig. 1 This is aTc induction experiment of <a href="http://partsregistry.org/Part:BBa_K737039">K737039</a>. Production of spot42 is induced by aTc. The translation of galK::GFP will be inhibited by spot42 in our expectation. And experimental results show that aTc induction works well for inhibition of GFP expression which is represented with decreasing RFU.<br />
</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:87px;" src="https://static.igem.org/mediawiki/2012/f/f9/Ouc-project-discussion1.jpg" /></a><br />
<br/><p style="text-align:center; font-size:90%;">Fig. 2 This is experimental control for IPTG induction to verify whether IPTG induction work as expected. Because of the deletion of Plac, IPTG won’t exert effect to spot42 production which means that spot42 will be constitutively expressed at relatively high levels which repress expression of GFP. To compare with Fig. 1, it is obvious that spot42 gives approximately 2-fold repression rate of galK::GFP expression.</p><br />
<br/><br />
<br/><br />
<a><img style="margin-left:81px;" src="https://static.igem.org/mediawiki/2012/7/73/Ouc-project-discussion2.jpg" /></a><br />
<br/><p style="text-align:center; font-size:90%;">Fig. 3 The GFP expression in all genetic circuits without any induction is shown. <a href="http://partsregistry.org/Part:BBa_K737051">K737051</a> is lacI-repressed GFP generator. R0011-E0240 is PlacI GFP generator without tetR repression. <a href="http://partsregistry.org/Part:BBa_K737038">K737038</a> is IPTG-inducible galK::GFP generator without IPTG induction. <a href="http://partsregistry.org/Part:BBa_K737040">K737040</a> has been mentioned in Figure 2. <br/><br />
</p><br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/1/1e/Ouc-project-designmaking-discussion4.jpg" /></a><br />
<br/><p style="text-align:center; font-size:90%;">Fig.4 aTc induction experiment of <a href="http://partsregistry.org/Part:BBa_K737041">BBa_K737041</a> is shown in (A). IPTG induction experiment of <a href="http://partsregistry.org/Part:BBa_K737040">BBa_K737040</a> is shown in (B). Both of them serve as the experimental control for comparators and ratio sensors. Two genetic circuits when separately introduced into cells won’t respond to the inducers for LacI generator assembled with Ptet while tetR generator with Plac. The experimental results meet our expectation and pose no negative effect to GFP expression. Besides, high concentration of aTc induction to <a href="http://partsregistry.org/Part:BBa_K737039">K737039</a> is closed to the result of (A), which indicates the effects of aTc induction and repression of spot42 to GFP expression.</p><br/><br />
<br/><br />
<a><img style="margin-left:70px;" src="https://static.igem.org/mediawiki/2012/1/11/Ouc-project-designmaking-discussion5.jpg" /></a><br />
<br/><p style="text-align:center; font-size:90%;">Fig.5 (A) is IPTG induction experiment of <a href="http://partsregistry.org/Part:BBa_K737052">K737052</a> which is aTc-inducible GFP generator. The genetic circuit is involved in the low copy plasmid pSB4A5 .<br />
(B) the experimental control.<br />
We can clearly to see that aTc induction of spot42 works perfectly well compared to (B).<br />
<br />
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