Difference between revisions of "Team:RDFZ-China/Demonstrate"

 
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                     <ul>
 
                     <ul>
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                         <li><a href="#section1">Overall?</a></li>
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                         <li><a href="#section1">Overall</a></li>
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                         <li><a href="#section2">Predecessors</a></li>
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                         <li><a href="#section2">Lethal Gene</a></li>
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                         <li><a href="#section3">Project Xscape</a></li>
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                         <li><a href="#section3">Thermal Regulator</a></li>
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                         <li><a href="#section4">For Fermentation</a></li>
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                         <li><a href="#section4">Density Regulator</a></li>
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                         <li><a href="#section5">For Therapy</a></li>
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                         <li><a href="#section5">Integrase</a></li>
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                         <li><a href="#section6">Metabolic Stress</a></li>
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                         <li><a href="#section6">Therapeutic Bacteria</a></li>
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                         <li><a href="#section7">DIY Bio and Biosafety</a></li>
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                         <li><a href="#section7">Capacity Monitor</a></li>
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                        <li><a href="#section8">Community and Future</a></li>
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                        <li><a href="#section9">References</a></li>
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         <div id="comic">
 
         <div id="comic">
 
             <p>
 
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                <img src="https://static.igem.org/mediawiki/2018/thumb/1/18/T--RDFZ-China--PJDhead1263620.jpg/800px-T--RDFZ-China--PJDhead1263620.jpg" alt="pdcomic" />
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              <img src="https://static.igem.org/mediawiki/2018/c/c3/T--RDFZ-China--Result-page.png" alt="pdcomic" />
 
             </p>
 
             </p>
 
         </div>
 
         </div>
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         <div class="Overall">
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         <div class="description">
 
             <div class="topic-title" id="section1">
 
             <div class="topic-title" id="section1">
 
                 <h3>Overall</h3>
 
                 <h3>Overall</h3>
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                 <p>✔Our lethal genes are functional, the DNase can degrade genome.  
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                 <p>✔Our lethal genes are functional, the DNase can degrade genome. </p>
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                  ✔A serie of thermal regulators work as we expected, the switch can either work when we turn it from on to off and off to on, also it can convert the positive regulation to negative regulation through an additional repressor:sRNA
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                <p>✔A serie of thermal regulators work as we expected, the switch can either work when we turn it from on to off and off to on, also it can convert the positive regulation to negative regulation through an additional repressor: sRNA</p>
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                  ✔Integrase can be used as initiator for the device
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                <p>✔Density Regulator work as expected, they can convert the positive regulation to negative regulation through an additional repressor: PhlF</p>
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                  ✔Density Regulator work as expected, they can convert the positive regulation to negative regulation through an additional repressor: PhlF
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                <p>✔Integrase can be used as initiator for the device</p>
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                  ✔Drug’s release can be thermal regulated
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                <p>✔Drug’s release can be thermal regulated</p>
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</p>
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             </div>
 
             </div>
 
             <div class="topic-title" id="section2">
 
             <div class="topic-title" id="section2">
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                 <h3>Predecessors</h3>
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                 <h3>Lethal Gene</h3>
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                 <p>For decades, researchers were striving to build biosafety devices through auxotrophy or external inducive kill switches(Lee et al.); of the latter holins and restriction enzymes are most commonly used. Most failures of the previous devices were due to mutations and evolution of immunity(Moe-Behrens et al.) . </p>
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                <h4>-clearness</h4>
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                 <p>The two major threats of engineered microbes’ leakage are the possible horizontal gene transfer which will lead to the spread of recombinant DNA to the ecosystem, or the fact that engineered bacteria could contaminate or overrun the natural habitat.(Wright et al.))</p>
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                 <p>ccdB colony was picked from the plate and transfer to the bacterium tube adding 10^3M and 10^2 M of IPTG to M9 medium including Cm antibiotic, tubes are cultured overnight, 37 degree Celsius and 200 rpm. </p>
 +
                <img src="https://static.igem.org/mediawiki/2018/thumb/2/2e/T--RDFZ-China--ccdBqualitative.jpg/800px-T--RDFZ-China--ccdBqualitative.jpg" width="100%">
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                <b class="text-comment">Fig.1 Differences between induced and uninduced ccdB broth.</b>
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                <p>Visible difference can be seen between non-induced and the induced ccdB, so we can qualitatively conclude the ccdB works as expected.</p>
 +
                 <p>Since miniColicin was hard to construct, we changed our order several times and obtained a plasmid with missing RBS and an extra integration mutation behind the start codon, so we perform the characterization exam quite roughly.</p>
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                <img src="https://static.igem.org/mediawiki/2018/thumb/6/63/T--RDFZ-China--mE2quali.jpg/800px-T--RDFZ-China--mE2quali.jpg" width="100%">
 +
                <b class="text-comment">Fig.2 Rough characterization of miniColicin’s lethality in DWP.</b>
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                <p>But still, the ccdB broth has left in the well for 2 days long, and miniColicin had stayed in for 1 day, the well with IPTG added showed visible clearness, which also indicates it is functional as a lethal gene.</p>
 +
                <h4>-cfu</h4>
 +
                <p>We carried out the cell forming unit exam for BBa_K2572019, by adding 100ul uninduced broth to the petri dish with or without IPTG. </p>
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                <img src="https://static.igem.org/mediawiki/2018/thumb/8/85/T--RDFZ-China--cfu.jpg/800px-T--RDFZ-China--cfu.jpg" width="100%">
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                <b class="text-comment">Fig3 a) Plates with broth dilution factor of 5, plates do contain 1E-3M IPTG. With colony forming unit 546, 585, 622<br>Fig3 b) Plates with broth dilution factor of 5, plates do not contain IPTG. With colony forming unit 1448, 2661, 1778.</b>
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                <p>There is about 70% reduction of induced colony formed compare to the non-induced ones, so we can say this device is functional properly. We assumed that if the induction started inside the tube, the cfu will drop dramatically.</p>
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 +
                <h4>-DNA cleavage</h4>
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                <img src="https://static.igem.org/mediawiki/parts/9/9d/T--RDFZ-China--GPT.png" width="100%">
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                <b class="text-comment">Fig.4 The DNA cleavage image from genscript.</b>
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                <p>The problem with Nucleases are that it might leak during synthesis or subclone construction, so our plasmid came very very late. The assay was carried out by genescript technicians during our subclone preparation. In the graph, DNA is quite completely degraded, which means our part worked as expected.</p>
 
             </div>
 
             </div>
 
             <div class="topic-title" id="section3">
 
             <div class="topic-title" id="section3">
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                 <h3>Project Xscape</h3>
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                 <h3>Thermal Regulator</h3>
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                <p>Under this circumstance, this year we decided to be a fundamentalist to synthetic biology, using genetic circuits and logic gates to establish biosafety devices which can apply to real-world situations.</p>
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                            <h4>-can it work</h4>
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                <p>Since cell death and lysis mean there is a continual presence of free DNA in the environment, holins, which are most widely used are excluded from our choices, and colicin E2 nucleases (Darmstadt iGEM2016) came into our site. We choose site non-specific nucleases since the entire genome and plasmids needed to be entirely digested to prevent the spread, and we use nucleases from a different family to prevent the possible evolution of nuclease inhibitors. Artificial DNA, RNA, and amino acids are a good solution, but due to its high cost so far, it is not applicable to most of the user.</p>
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                            <p>First, we characterize the Thermal Sensitive Regulators, to see if they work. We simply put them into 37 degree Celsius shaker and 42 degree Celsius shaker, and see if they glow as we expected.</p>
 +
                            <img src="https://static.igem.org/mediawiki/parts/thumb/7/79/T--RDFZ-China--4237.jpg/1200px-T--RDFZ-China--4237.jpg" width="100%">
 +
                            <p>K2572000 is TlpA39, which was selected out with the same method as TlpA36, we thought that 39 degree might be a good temperature for human, which was not too high to hurt people, also not too low to mixed up with others. K2572001 is Tcl42, quite similar with TlpA protein and its originated from bacteriophage, which with regulation system cl repressor. </p>
 +
                            <p>However, we found that Tcl38 is not working as we expected, and after sequencing, we were quite sure that the reporter gene was lost. So, in the following characterization, Tcl38 was used as a negative control.</p>
 +
                            <h4>-as we expected</h4>
 +
                            <p>Then we performed the characterization under different temperature. Petri dishes were placed into incubators with different temperatures and grew for 24 hours.</p>
 +
                            <img src="https://static.igem.org/mediawiki/2018/thumb/8/8e/T--RDFZ-China--TSRquali.jpeg/800px-T--RDFZ-China--TSRquali.jpeg" width="100%">
 +
                            <p>We can see that at 35 degree Celsius, TlpA36 starts to derepress. At 37 degree Celsius, TlpA39 starts to derepress. At 39.5 degree Celsius, Tcl42 starts to express. Leakage was observed, transcription of pTlpA was initiated below the expected temperature. ETH Zurich iGEM2017 improved this by simply increase the expression of TlpA protein, since they assumed that the leakage was caused by lack of repression.</p>
  
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            </div>
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                            <h4>We used plate reader to characterize this set of Thermal Regulators, they were incubicated under different temperature and transformed to plate reader after 18 hours.</h4>
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            <div class="topic-title" id="section4">
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                            <img src="https://static.igem.org/mediawiki/2018/4/42/T--RDFZ-China--TSR-Quantitative.png" width="100%">
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                <h3>For fermentation</h3>
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                            <b class="text-comment">This fig. shows the expression of thermal regulators under 30 33 36 39 42 45 degree Celsius,we can see the leakage is low, unlike what we have seen on the petri dishes.</b>
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                <p>The first device we build is for the fermentation; we want to execute any escaped engineered bacteria from the fermenter, whether the escape happened accidentally or intentionally. We used two environment factors to monitor the bacteria’s situation: temperature and population density, thet are both high and tunable in the fermenter, so our device will initiate when temperature and density are both low. We used thermal sensitive regulator (NUS iGEM2017)(Piraner et al.) and quorum sensing regulator (MIT iGEM2004) (Canton et al.)as our sensor, with sRNA(Storz et al.) and tetR family repressor PhlF(Glasgow iGEM2015)(Stanton et al.) as the signal inverter. We added an integrase (Peking iGEM2017) controlled by the thermal sensitive regulator which will enable the promoter of a lethal gene to potentially express when temperature rise in the fermenter so that bacteria can survive at the very beginning. Also, we built a model to stimulate the minimum autoinducer required at the beginning of the fermentation, same as the purpose of integrase. This model is for keeping bacteria alive at the very beginning of fermentation. Together they form a NOR gate that will lead to cell death through genome degradation when both temperature and density decrease.</p>
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            </div>
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            <div class="topic-title" id="section5">
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                <h3>For Therapy</h3>
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                <p>The second device we build is for therapeutic bacteria. The device can carry out noninvasive tracing through ultrasound imaging of the gas vesicle(Shapiro et al.), release the drug (from SHSBNU 2017) controlled by a thermosensitive regulator at nidus by ultrasound tissue heating, and heat to a higher temperature to release nuclease and kill the bacteria after it finishes its mission. </p>
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            </div>
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            <div class="topic-title" id="section6">
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                <h3>For Metabolic Stress</h3>
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                <p>We applied capacity monitor (Ceroni et al.) to quantify the expression burden of all our systems, and to reduce the metabolic stress, we designed another device for fermentation which used a LuxR repressive promoter (Peking iGEM2011) and cold-regulated 5’UTR region (Ionis Paris 2017). This device only involves one transcriptional regulator, which will be less energy consuming. </p>
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            </div>
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            <div class="topic-title" id="section7">
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                <h3>DIY bio and Biosafey</h3>
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                <p>Back to the growing and glowing synthetic biology community, despite the current majority doing it on campus, more and more people are starting it at home, and they call themselves Genehacker or DIY biologists. The lack of sufficient training and efficient surveillance will be a time bomb for which we do know that monstrous harmful bioproducts will be made someday in the future, and indeed, it will be a significant threat to the current biosafety basis. Recall our memory to iGEM2009, Peking surveyed DIY bio, almost ten years later, we conducted a similar DIY bio-survey. We tried to order materials for molecular experiments, using the delivery address to our home, and the result was quite shocking that we can buy almost everything for the molecular experiment, from the internet. Then, we went through relevant laws and regulations throughout the world, in which we found no laws related to credit certification and the address certification about the people who book the biology reagent. Most of the laws are about the quality certification and how they would serve the user after they bought this. We interviewed the Director of a center for disease control and prevention. He said that in his experiment with the disease caused by the bacteria leak, vast impacts such as environmental pollution had been observed. and our country has been making great effort towards elimenating such leakage accidents and incidents. He said it is not easy to solve the problem with hard work, rather it needs the cooperation between all the countries. He made an example of 731 army during the second world war two, in which the outbreak of pathogens caused significant physical and social harm. We are still on our way to win the battle, and the effort still needs to be put in.</p>
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            </div>
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            <div class="topic-title" id="section8">
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                <h3>Community and Future</h3>
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                <p>Also, we hosted two major meeting in Beijing. In a biosafety forum in October, we invited a team leader who runs his high school lab, lab teachers from a university lab, and a former team member from Peking iGEM2009, who participated in their DIY bio investigation ten years ago.</p>
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                <p>We concluded that the development of DIY bio should be taken seriously, and the permanent way to solve its safety and security concerns is through implanting biosafety awareness into our academic culture. Also, as iGEMers, we should strive to be the considerate and responsible leaders in our community, to ensure the biosafety issue has been taken properly. Another meeting was with biology Olympians all over China, in which we discussed the future of biology community during the meeting, especially with more and more high school iGEM teams coming up in China contrasted with the lack of relevant instruction and education to the students. We came up with the idea of setting up a collaboration between schools to share and overcome difficulties hand in hand. This kind of meeting will be continued after iGEM2018, since the community usually grows fast after every iGEM season. </p>
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                <p>Hopefully, years later, biosafety awareness and considerations can be seriously taken in communities, laboratory studies, and real-world applications.</p>
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            </div>
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        </div>
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        <div class="reference">
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            <div class="topic-title" id="section9">
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                <h3>References</h3>
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                <p>Canton, Barry, et al. “Refinement and Standardization of Synthetic Biological Parts and Devices.” Nature Biotechnology, vol. 26, no. 7, 2008, pp. 787–93, doi:10.1038/nbt1413.</p>
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−
                <p>Ceroni, Francesca, et al. “Quantifying Cellular Capacity Identifies Gene Expression Designs with Reduced Burden.” Nature Methods, vol. 12, no. 5, 2015, pp. 415–18, doi:10.1038/nmeth.3339.</p>
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−
                <p>Lee, Jeong Wook, et al. “Next-Generation Biocontainment Systems for Engineered Organisms.” Nature Chemical Biology, Springer US, 2018, p. 1, doi:10.1038/s41589-018-0056-x.</p>
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−
                <p>Moe-Behrens, Gerd H. G., et al. “Preparing Synthetic Biology for the World.” Frontiers in Microbiology, vol. 4, no. JAN, 2013, pp. 1–10, doi:10.3389/fmicb.2013.00005.</p>
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                <p>Piraner, Dan I., et al. “Tunable Thermal Bioswitches for in Vivo Control of Microbial Therapeutics.” Food, Pharmaceutical and Bioengineering Division 2017 - Core Programming Area at the 2017 AIChE Annual Meeting, vol. 2, no. November, Nature Publishing Group, 2017, pp. 695–702, doi:10.1038/nchembio.2233.</p>
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                <p>Shapiro, Mikhail G., et al. “Biogenic Gas Nanostructures as Ultrasonic Molecular Reporters.” Nature Nanotechnology, vol. 9, no. 4, Nature Publishing Group, 2014, pp. 311–16, doi:10.1038/nnano.2014.32.</p>
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                <p>Stanton, Brynne C., et al. “Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates.” Nature Chemical Biology, vol. 10, no. 2, 2014, pp. 99–105, doi:10.1038/nchembio.1411.</p>
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                <p>Storz, Gisela, et al. “Regulation by Small RNAs in Bacteria: Expanding Frontiers.” Molecular Cell, vol. 43, no. 6, 2011, pp. 880–91, doi:10.1016/j.molcel.2011.08.022.</p>
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                <p>Synthetic, How, and Biology Will. “Regenesis: How Synthetic Biology Will Reinvent Nature and Ourselves.” Choice Reviews Online, 2013, doi:10.5860/CHOICE.50-3835.</p>
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−
                <p>Wright, Oliver, et al. “Building-in Biosafety for Synthetic Biology.” Microbiology (United Kingdom), vol. 159, no. PART7, 2013, pp. 1021–35, doi:10.1099/mic.0.066308-0.</p>
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            </div>
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        </div>
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                            <h4>We verified that Thermal Regulators can turn from on to off. Most of the user was using turn on, but seldom people try to turn it off. We cultivated TlpA36 from high 37 degree Celsius to 30 degree Celsius, and measure it on plate reader</h4>
 +
                            <img src="https://static.igem.org/mediawiki/parts/6/61/T--RDFZ-China--TSRREV.png" width="100%">
 +
                            <b class="text-comment">This fig indicated that the expression stop as soon as it is being transformed from 37 degree to 30 degree, we concluded that the remaining fluorescent was from the undegraded GFP.</b>
 +
 
 +
                            <h4>Invert the signal by sRNA</h4>
 +
                            <p>We performed the thermal regulation characterization by co-transform our sensor plasmid (TlpA36-pTlpA-sRNA) and reporter plasmid(pTac-RiboJ-J61101-mNeonGreen). Then, they were incubated in 37°C and 30°C, for 20 hours. First, we performed a qualitative assay, by comparing the brightness of the broth.</p>
 +
                            <img src="https://static.igem.org/mediawiki/parts/thumb/d/d1/T--RDFZ-China--sRNAall.png/1200px-T--RDFZ-China--sRNAall.png" width="100%">
 +
                            <b class=" text-comment">The result showed no significanlty reduce in eexpression, it may be casued by the shortness of our seed region which is for inhibition</b>
 +
                        </div>
 +
                        <div class="topic-title" id="section4">
 +
                            <h3>Density Regulator</h3>
 +
                            <h4>-can it work</h4>
 +
                            <p>In order to demonstrate the density-regulated sensor is well controlled by the concentration of signal molecules (acyl-homoserine lactone, or AHLs) produced, we characterized the quorum sensing system (Lux) with purchased AHL molecules. Different concentrations of AHL were used to induce the transcription of sfGFP, attempting to identify the threshold concentration that turns on the transcription downstream pLux.</p>
 +
                            <p>AHL stocks in the concentration of 10<sup>-3</sup> M to 10<sup>-14</sup> M was prepared by serial dilutions. Then the AHL stocks were added into M9 media culture thousand-fold in volume to obtain M9 with AHL of concentrations ranging from 10<subp>-6</subp> M to 10<sup>-14</sup> M. IPTG was added into M9 to induce the expression of LuxR. </p>
 +
                            <img src="https://static.igem.org/mediawiki/2018/thumb/4/4d/T--RDFZ-China--PRV.jpg/800px-T--RDFZ-China--PRV.jpg" width="100%">
 +
                            <p>We first did a rough qualitative experiment to make sure this device is functional.</p>
 +
                            <p>From this figure we can see the device is functional.</p>
 +
                            <h4>-quantitatively</h4>
 +
                            <img src="https://static.igem.org/mediawiki/2018/2/28/T--RDFZ-China--Lux-characterization.png" width="100%">
 +
                            <p>The fluorescent intensity increased dramatically at the concentration of 10<subp>-8</subp> M, indicating the threshold concentration of AHL for Lux system is around 10<sup>-8</sup> M. At least 10<sup>-7</sup> M to 10<sup>-8</sup> M of AHL is required to activate the expression under the regulation of pLux.</p>
 +
                            <h4>-and reverse the signal</h4>
 +
                            <p>As mentioned before, we need to convert the signal to a negative response, which we accomplished by adding a PhlF repressor, combining to form part K2572016. PhlF binds to an operator in the pPhlF region to repress the expression of downstream suicide sequences (ccdB/GBSV1/colicin E2). When temperature and cell density are high, regulator PhlF (and sRNA) is produced to repress the expression of suicide genes. As temperature drops and community density decreases, simulating that engineered strains escape from the fermentation condition, PhlF is repressed and consequently turns on the expression of cytotoxic sequences.</p>
 +
                            <img src="https://static.igem.org/mediawiki/2018/2/2b/T--RDFZ-China--Lux%2BPhlF_characterization.png" width="100%">
 +
                            <p>Also a model was built to predict the function of this device. </p>
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                        </div>
 +
                        <div class="topic-title" id="section5">
 +
                            <h3>Integrase</h3>
 +
                            <p>The second device we build is for therapeutic bacteria. The device can carry out noninvasive tracing through ultrasound imaging of the gas vesicle(Shapiro et al.), release the drug (from SHSBNU 2017) controlled by a thermosensitive regulator at nidus by ultrasound tissue heating, and heat to a higher temperature to release nuclease and kill the bacteria after it finishes its mission. </p>
 +
                            <h4>-can it work</h4>
 +
                            <p>Quantitative Characterization of integrase was not as expected . We gain access to the integrase Bxb1 from 2017 Peking University iGEM team, who constructed a time-sequential logic system with the use of integrase (Peking iGEM2017). The integrase Bxb1 is regulated by the upstream pBAD operon, which activates in the presence of arabinose. </p>
 +
                            <p>Theoretically, presence of arabinose will turn on PBAD thus initiates the production of Bxb1. Bxb1 flips the promoter upstream of GFP and starts the transcription of GFP. Conversely, without arabinose induction, no green fluorescence should be observed in bacteria colonies. We assume this was due to the leakage of BAD operator. </p>
 +
                            <img src="https://static.igem.org/mediawiki/2018/7/71/T--RDFZ-China--Ara.jpg" width="100%">
 +
                            <b class="text-comment">From the fig, there are differences between the one with bxb1 plasmid and the ones without, they glows as expected with their combination. However, as the expression should increase with higher mole of arabinose, the experimental results showed that the expression was decreased.<br>The plasmids co-transformed were extracted out for sequencing, so we can not assess it right away.</b>
 +
                        </div>
 +
                        <div class="topic-title" id="section6">
 +
                            <h3>Therapeutic Bacteria</h3>
 +
                            <h4>-TlpA39-Vio</h4>
 +
                            <p>We constructed the pTlpA-VioABDE-TlpA39, and cultured it under different temperature. </p>
 +
                            <img src="https://static.igem.org/mediawiki/parts/thumb/7/7d/T--RDFZ-China--VioChara.jpeg/1200px-T--RDFZ-China--VioChara.jpeg" width="100%">
 +
                            <b class="text-comment">The fig shows that there was leakage of PVA, which was the molecule produced by VioABDE.</b>
 +
 
 +
                        </div>
 +
                        <div class="topic-title" id="section7">
 +
                            <h3>Capacity Monitor</h3>
 +
                            <p>We constructed bacteria with either GFP or VioABDE, and with both of them to monitor the capacity of gene expression. The three bacteria strains were cultured for 12 hours with green fluorescence and OD constantly monitored. </p>
 +
                            <img src="https://static.igem.org/mediawiki/2018/2/20/T--RDFZ-China--CM%2BVio.png" width="100%">
 +
                            <b class="text-comment">From the fig, by comparing the fluorescence of GFP-containing E. coli, the fluorescence intensity rose faster and higher for bacteria without VioABDE expression than E. coli with VioABDE inserted and expressed. It signifies that VioABDE expression consumed notable resources in the host cell and caused expression burden in the cell. The fluorescence of VioABDE shows that VioABDE does not emit green fluorescence thus not significantly influencing the results obtained.</b>
 +
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                        <br>
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Latest revision as of 03:35, 18 October 2018

pdcomic

Overall

✔Our lethal genes are functional, the DNase can degrade genome.

✔A serie of thermal regulators work as we expected, the switch can either work when we turn it from on to off and off to on, also it can convert the positive regulation to negative regulation through an additional repressor: sRNA

✔Density Regulator work as expected, they can convert the positive regulation to negative regulation through an additional repressor: PhlF

✔Integrase can be used as initiator for the device

✔Drug’s release can be thermal regulated

Lethal Gene

-clearness

ccdB colony was picked from the plate and transfer to the bacterium tube adding 10^3M and 10^2 M of IPTG to M9 medium including Cm antibiotic, tubes are cultured overnight, 37 degree Celsius and 200 rpm.

Fig.1 Differences between induced and uninduced ccdB broth.

Visible difference can be seen between non-induced and the induced ccdB, so we can qualitatively conclude the ccdB works as expected.

Since miniColicin was hard to construct, we changed our order several times and obtained a plasmid with missing RBS and an extra integration mutation behind the start codon, so we perform the characterization exam quite roughly.

Fig.2 Rough characterization of miniColicin’s lethality in DWP.

But still, the ccdB broth has left in the well for 2 days long, and miniColicin had stayed in for 1 day, the well with IPTG added showed visible clearness, which also indicates it is functional as a lethal gene.

-cfu

We carried out the cell forming unit exam for BBa_K2572019, by adding 100ul uninduced broth to the petri dish with or without IPTG.

Fig3 a) Plates with broth dilution factor of 5, plates do contain 1E-3M IPTG. With colony forming unit 546, 585, 622
Fig3 b) Plates with broth dilution factor of 5, plates do not contain IPTG. With colony forming unit 1448, 2661, 1778.

There is about 70% reduction of induced colony formed compare to the non-induced ones, so we can say this device is functional properly. We assumed that if the induction started inside the tube, the cfu will drop dramatically.

-DNA cleavage

Fig.4 The DNA cleavage image from genscript.

The problem with Nucleases are that it might leak during synthesis or subclone construction, so our plasmid came very very late. The assay was carried out by genescript technicians during our subclone preparation. In the graph, DNA is quite completely degraded, which means our part worked as expected.

Thermal Regulator

-can it work

First, we characterize the Thermal Sensitive Regulators, to see if they work. We simply put them into 37 degree Celsius shaker and 42 degree Celsius shaker, and see if they glow as we expected.

K2572000 is TlpA39, which was selected out with the same method as TlpA36, we thought that 39 degree might be a good temperature for human, which was not too high to hurt people, also not too low to mixed up with others. K2572001 is Tcl42, quite similar with TlpA protein and its originated from bacteriophage, which with regulation system cl repressor.

However, we found that Tcl38 is not working as we expected, and after sequencing, we were quite sure that the reporter gene was lost. So, in the following characterization, Tcl38 was used as a negative control.

-as we expected

Then we performed the characterization under different temperature. Petri dishes were placed into incubators with different temperatures and grew for 24 hours.

We can see that at 35 degree Celsius, TlpA36 starts to derepress. At 37 degree Celsius, TlpA39 starts to derepress. At 39.5 degree Celsius, Tcl42 starts to express. Leakage was observed, transcription of pTlpA was initiated below the expected temperature. ETH Zurich iGEM2017 improved this by simply increase the expression of TlpA protein, since they assumed that the leakage was caused by lack of repression.

We used plate reader to characterize this set of Thermal Regulators, they were incubicated under different temperature and transformed to plate reader after 18 hours.

This fig. shows the expression of thermal regulators under 30 33 36 39 42 45 degree Celsius,we can see the leakage is low, unlike what we have seen on the petri dishes.

We verified that Thermal Regulators can turn from on to off. Most of the user was using turn on, but seldom people try to turn it off. We cultivated TlpA36 from high 37 degree Celsius to 30 degree Celsius, and measure it on plate reader

This fig indicated that the expression stop as soon as it is being transformed from 37 degree to 30 degree, we concluded that the remaining fluorescent was from the undegraded GFP.

Invert the signal by sRNA

We performed the thermal regulation characterization by co-transform our sensor plasmid (TlpA36-pTlpA-sRNA) and reporter plasmid(pTac-RiboJ-J61101-mNeonGreen). Then, they were incubated in 37°C and 30°C, for 20 hours. First, we performed a qualitative assay, by comparing the brightness of the broth.

The result showed no significanlty reduce in eexpression, it may be casued by the shortness of our seed region which is for inhibition

Density Regulator

-can it work

In order to demonstrate the density-regulated sensor is well controlled by the concentration of signal molecules (acyl-homoserine lactone, or AHLs) produced, we characterized the quorum sensing system (Lux) with purchased AHL molecules. Different concentrations of AHL were used to induce the transcription of sfGFP, attempting to identify the threshold concentration that turns on the transcription downstream pLux.

AHL stocks in the concentration of 10-3 M to 10-14 M was prepared by serial dilutions. Then the AHL stocks were added into M9 media culture thousand-fold in volume to obtain M9 with AHL of concentrations ranging from 10-6 M to 10-14 M. IPTG was added into M9 to induce the expression of LuxR.

We first did a rough qualitative experiment to make sure this device is functional.

From this figure we can see the device is functional.

-quantitatively

The fluorescent intensity increased dramatically at the concentration of 10-8 M, indicating the threshold concentration of AHL for Lux system is around 10-8 M. At least 10-7 M to 10-8 M of AHL is required to activate the expression under the regulation of pLux.

-and reverse the signal

As mentioned before, we need to convert the signal to a negative response, which we accomplished by adding a PhlF repressor, combining to form part K2572016. PhlF binds to an operator in the pPhlF region to repress the expression of downstream suicide sequences (ccdB/GBSV1/colicin E2). When temperature and cell density are high, regulator PhlF (and sRNA) is produced to repress the expression of suicide genes. As temperature drops and community density decreases, simulating that engineered strains escape from the fermentation condition, PhlF is repressed and consequently turns on the expression of cytotoxic sequences.

Also a model was built to predict the function of this device.

Integrase

The second device we build is for therapeutic bacteria. The device can carry out noninvasive tracing through ultrasound imaging of the gas vesicle(Shapiro et al.), release the drug (from SHSBNU 2017) controlled by a thermosensitive regulator at nidus by ultrasound tissue heating, and heat to a higher temperature to release nuclease and kill the bacteria after it finishes its mission.

-can it work

Quantitative Characterization of integrase was not as expected . We gain access to the integrase Bxb1 from 2017 Peking University iGEM team, who constructed a time-sequential logic system with the use of integrase (Peking iGEM2017). The integrase Bxb1 is regulated by the upstream pBAD operon, which activates in the presence of arabinose.

Theoretically, presence of arabinose will turn on PBAD thus initiates the production of Bxb1. Bxb1 flips the promoter upstream of GFP and starts the transcription of GFP. Conversely, without arabinose induction, no green fluorescence should be observed in bacteria colonies. We assume this was due to the leakage of BAD operator.

From the fig, there are differences between the one with bxb1 plasmid and the ones without, they glows as expected with their combination. However, as the expression should increase with higher mole of arabinose, the experimental results showed that the expression was decreased.
The plasmids co-transformed were extracted out for sequencing, so we can not assess it right away.

Therapeutic Bacteria

-TlpA39-Vio

We constructed the pTlpA-VioABDE-TlpA39, and cultured it under different temperature.

The fig shows that there was leakage of PVA, which was the molecule produced by VioABDE.

Capacity Monitor

We constructed bacteria with either GFP or VioABDE, and with both of them to monitor the capacity of gene expression. The three bacteria strains were cultured for 12 hours with green fluorescence and OD constantly monitored.

From the fig, by comparing the fluorescence of GFP-containing E. coli, the fluorescence intensity rose faster and higher for bacteria without VioABDE expression than E. coli with VioABDE inserted and expressed. It signifies that VioABDE expression consumed notable resources in the host cell and caused expression burden in the cell. The fluorescence of VioABDE shows that VioABDE does not emit green fluorescence thus not significantly influencing the results obtained.

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